Abstract:

Objects of the present invention are to provide a method for designing an
optimum delivery preparation with the use of a convenient experiment
and/or assay and to search for the thus produced delivery vehicle. The
present invention provides a method for producing a delivery vehicle for
achieving the delivery of a desired substance to a desired site, which
comprises the steps of: A) measuring in vitro affinity of candidate
delivery vehicles for a cell surface molecule such as a lectin associated
with the site; and B) selecting a delivery vehicle having in vitro
affinity corresponding to delivery to the desired site.

Claims:

1.-111. (canceled)

112. A delivery vehicle for achieving delivery to a desired site in which
a strong binding IC involved in in vitro affinity for a cell surface
molecule associated with the desired site is low and a weak binding IC
involved in in vitro affinity for the same is high, and the cell surface
molecule is selected from the group consisting of a lectin, an adhesion
molecule, an integrin, an immunoglobulin, a sialomucin, a cadherin, a
protein, a lipid, a receptor, an antigen, an enzyme, a metalloprotease, a
tyrosine phosphatase, a glycolipid, a glycoprotein, a proteoglycan, a
costimuratory molecule, a membrane protein, and an extracellular matrix.

113. The delivery vehicle according to claim 112, in which the delivery
vehicle is a liposome, and the boundary between the strong binding IC and
the weak binding IC is between 30 and 31 for "n" of ICn.

114. The delivery vehicle according to claim 113, in which the delivery
vehicle is a sugar-chain-modified liposome.

115. The delivery vehicle according to claim 112, in which the desired
site is selected from the group consisting of an inflammation site and a
cancer site.

116. The delivery vehicle according to claim 112, in which an inhibitory
concentration at a strong binding IC which is approximately IC30 or less
is 10.sup.-9M or less and an inhibitory concentration at a weak binding
IC which is approximately IC31 or more is 10.sup.-9M or more as to in
vitro affinity for a lectin associated with a desired state.

117. The delivery vehicle according to claim 112, which satisfies at least
one condition selected from the group consisting of a condition that the
inhibitory concentration at IC 10 is 10.sup.-9M or less, a condition that
an inhibitory concentration at IC20 is 10.sup.-9M or less and a condition
that an inhibitory concentration at IC30 is 10.sup.-9M or less for the
strong binding IC as to in vitro affinity for a lectin associated with a
desired site, and satisfies at least one condition selected from the
group consisting of a condition that the inhibitory concentration at IC40
is 10.sup.-9M or more, a condition that the inhibitory condition of IC50
is 10.sup.-9M or more and a condition that the inhibitory concentration
at IC60 is 10.sup.-9M or more for the weak binding IC as to in vitro
affinity for a lectin associated with a desired site.

118. The delivery vehicle according to claim 112, in which the IC is
measured based on affinity for E-selectin.

121. A method for producing a delivery vehicle for achieving the delivery
of a desired substance to a desired site, which comprises the steps of:A)
measuring in vitro affinity of candidate delivery vehicles for a cell
surface molecule associated with the desired site; andB) selecting a
delivery vehicle having in vitro affinity corresponding to delivery to
the desired site,in which the cell surface molecule is selected from the
group consisting of a lectin, an adhesion molecule, an integrin, an
immunoglobulin, a sialomucin, a cadherin, a protein, a lipid, a receptor,
an antigen, an enzyme, a metalloprotease, a tyrosine phosphatase, a
glycolipid, a glycoprotein, a proteoglycan, a costimuratory molecule, a
membrane protein, and an extracellular matrix.

122. The method according to claim 121, which further comprises the step
of causing the selected delivery vehicle to contain a substance to be
delivered.

123. The method according to claim 121, in which the delivery vehicle
contains a sugar-chain-modified liposome.

125. The method according to claim 121, in which the cell surface molecule
contains E-selectin and the site is selected from the group consisting of
a site of the liver, a site of the small intestine, a site of the large
intestine, a site of the lymph node, a site of the heart, a site of the
pancreas, a site of the lungs, a site of the brain, a site of the bone
marrow, a site in blood, a site of the kidney, a site of the spleen, a
site of the thymus gland, a site of muscle, an inflammation site, and a
cancer site.

126. The method according to claim 121, in which the affinity is
represented by n % inhibitory concentration (ICn), wherein "n" ranges
from 1 to 99.

127. The method according to claim 121, in which the measurement of
affinity comprises measurement at a strong binding IC that is
approximately IC30 or less and measurement at a weak binding IC that is
approximately IC31 or more.

128. The method according to claim 121, in which the measurement of
affinity comprises measurement at a strong binding IC that is at least
one point between IC30 and IC10 and measurement at a weak binding IC that
is at least one point between IC40 and IC60.

129. The method according to claim 121, in which the measurement of
affinity comprises measurement at a strong binding IC that is
approximately IC30 or less and a candidate having a low inhibitory
concentration at the strong binding IC is selected.

130. The method according to claim 121, in which the measurement of
affinity comprises measurement at a strong binding IC that is
approximately IC30 or less and a candidate having an inhibitory
concentration of 10.sup.-9M or less at the strong binding IC is selected.

131. The method according to claim 121, in which the measurement of
affinity comprises measurement at a strong binding IC that is
approximately IC30 or less and a candidate having a low inhibitory
concentration at the strong binding IC is selected, wherein the selection
is performed when at least one condition selected from the group
consisting of a condition that the inhibitory concentration at IC 10 is
10.sup.-9M or less, a condition that an inhibitory concentration at IC20
is 10.sup.-9M or less and a condition that an inhibitory concentration at
IC30 is 10.sup.-9M or less is satisfied.

132. The method according to claim 121, in which the measurement of
affinity comprises measurement at a weak binding IC that is approximately
IC31 or more and a candidate having a high inhibitory concentration at
the strong binding IC is selected.

133. The method according to claim 121, in which the measurement of
affinity comprises measurement at a weak binding IC that is approximately
IC31 or more and a candidate having an inhibitory concentration of
10.sup.-9M or more at the weak binding IC is selected.

134. The method according to claim 121, in which the measurement of
affinity comprises measurement at a weak binding IC that is approximately
IC31 or more and a candidate having a high inhibitory concentration at
the weak binding IC is selected, wherein the selection is performed when
at least one condition selected from the group consisting of a condition
that the inhibitory concentration at IC40 is 10.sup.-9M or more, a
condition that the inhibitory concentration at IC50 is 10.sup.-9M or more
and a condition that the inhibitory concentration at IC60 is 10.sup.-9M
or more is satisfied.

135. The method according to claim 121, in which the measurement of
affinity comprises measurement at a strong binding IC that is
approximately IC30 or less and a weak binding IC that is approximately
IC31 or more and a candidate having a low inhibitory concentration at the
strong binding IC and a high inhibitory concentration at the weak binding
IC is selected.

136. The method according to claim 121, in which the measurement of
affinity comprises measurement at a strong binding IC that is
approximately IC30 or less and a weak binding IC that is approximately
IC31 or more and a candidate having an inhibitory concentration of
10.sup.-9M or less at the strong binding IC and an inhibitory
concentration of 10.sup.-9M or more at the weak binding IC is selected.

137. The method according to claim 121, in which the measurement of
affinity comprises measurement at a strong binding IC that is
approximately IC30 or less and a weak binding IC that is approximately
IC31 or more and a candidate having a low inhibitory concentration at the
strong binding IC and a high inhibitory concentration at the weak binding
IC is selected,wherein the selection is performed when at least one
condition selected from the group consisting of a condition that the
inhibitory concentration at IC10 is 10.sup.-9M or less, a condition that
an inhibitory concentration at IC20 is 10.sup.-9M or less and a condition
that an inhibitory concentration at IC30 is 10.sup.-9M or less is
satisfied; and in terms of the weak binding IC, at least one condition
selected from the group consisting of a condition that the inhibitory
concentration at IC40 is 10.sup.-9M or more, a condition that the
inhibitory concentration at IC50 is 10.sup.-9M or more and a condition
that the inhibitory concentration at IC60 is 10.sup.-9M or more is
satisfied.

138. The method according to claim 121, in which the measurement of
affinity is performed by a method selected from the group consisting of a
competitive inhibition assay, a noncompetitive inhibition assay, and a
binding assay.

139. The method according to claim 121, in which the delivery vehicle
contains a sugar-chain-modified liposome and the analysis of the
composition comprises analysis of the sugar chain type and density of the
sugar-chain-modified liposome.

140. The method according to claim 121, in which a linker is used for the
modification of liposome.

141. The method according to claim 140, in which the linker is a protein.

142. The method according to claim 140, in which the linker is an albumin.

143. The method according to claim 121, which further comprises a step of
hydrophilizing the liposome.

144. A pharmaceutical composition, which contains a drug to be used for
prevention or treatment, and the delivery vehicle according to claim 112.

145. A pharmaceutical composition, which contains a drug to be used for
prevention or treatment, and a delivery vehicle that is produced by the
method according to claim 121.

146. A method for preventing or treating a subject who requires delivery
of a drug to a desired site, which comprises the steps of:A) measuring in
vitro affinity of candidate delivery vehicles, which are intended for
achieving delivery to a desired site, for a cell surface molecule
associated with the desired site;B) selecting a delivery vehicle having
in vitro affinity corresponding to delivery to the desired site; andC)
administering a drug required for prevention or treatment to the subject
with the use of the selected delivery vehicle,in which the cell surface
molecule is selected from the group consisting of a lectin, an adhesion
molecule, an integrin, an immunoglobulin, a sialomucin, a cadherin, a
protein, a lipid, a receptor, an antigen, an enzyme, a metalloprotease, a
tyrosine phosphatase, a glycolipid, a glycoprotein, a proteoglycan, a
costimuratory molecule, a membrane protein, and an extracellular matrix.

Description:

TECHNICAL FIELD

[0001]The present invention relates to the design of a remedy comprising
an optimum probe that is selected via in vitro experiments from among
probes that can be used as drug delivery systems for treatment (DDSs are
used for recognizing target cells and/or tissues such as cancer and then
locally delivering drugs or genes to affected parts) or as cell- and/or
tissue-sensing probes for diagnosis, a sugar-chain-modified liposome
produced with the use of such probe, and a liposome formulation in which
a drug, a gene, or the like is encapsulated, which are applicable to
medical and/or pharmaceutical fields in addition to the fields of
pharmaceutical products and cosmetics.

BACKGROUND ART

[0002]The National Nanotechnology Initiative (NNI) of the U.S.A. has set
up "a drug or gene delivery system (DDS: drug delivery system) for taking
a shot at cancer cells or target tissues," as an example of a specific
goal for realization. The Council for Science and Technology Policy in
Japan has set up "ultra small system material for medical use and
nanobiology for using and controlling biological mechanisms" as a
prioritized area of the Promotion Strategy for Nanotechnology and
Materials Area. One of the research and development objectives of the
Council over the next 5 years is "Establishment of Basic Seeds for
Technology Including Biofunctional Materials, Pinpoint Treatment, and the
like for Extending Healthy Life Spans." Meanwhile, incidences and
mortality of cancer are increasing yearly as the aging society emerges.
Development of a targeting DDS that is a novel material for treatment is
expected. Importance of targeting DDS nanomaterials without side effects
has attracted attention in regards to other diseases. The size of the
market is expected to exceed 10 trillion yen in the near future.
Moreover, these materials are also expected to be used for diagnosis in
addition to treatment.

[0003]A therapeutic effect of a pharmaceutical product is achieved when
the drug reaches a specific target site and then acts on the site. On the
other hand, a side effect resulting from a pharmaceutical product means
that a drug acts on undesired sites. Therefore, development of drug
delivery systems is desired for effective and safe use of drugs. In
particular, a targeting DDS is a concept whereby a drug is delivered to
"in vivo sites that need treatment," "in a necessary amount," "for a
required time length." Liposomes, which are microparticulate carriers,
have attracted attention as a typical material for use in such purposes.
Passive targeting methods that involve varying lipid type, composition
ratio, particle diameter, and surface charge of liposomes have been
attempted to impart targeting functions to such particles. However, these
methods are still insufficient and need further improvement.

[0004]Meanwhile, an active targeting method has also been attempted to
enable highly-functional targeting. This method is also referred to as
the "missile drug" and is an ideal targeting method. This method has not
yet been completed either at home or abroad, and the future development
thereof is greatly expected. This method comprises binding a ligand onto
a liposome membrane surface, so as to cause a receptor existing on the
cell membrane surfaces of a target tissue to specifically recognize the
ligand. Active targeting is made possible by the use of this method.
Possible ligands for such receptors existing on cell membrane surfaces to
be targeted by the active targeting method are antigens, antibodies,
peptides, glycolipids, glycoproteins, and the like. Of these, the fact
that sugar chains of glycolipids or glycoproteins play important roles as
information molecules in various cell-to-cell communications such as
development or morphological formation of living body tissue, cell
proliferation or differentiation, biophylaxis, the fertilization
mechanism, canceration, and the metastasis mechanism thereof is being
clarified.

[0006]Many studies have been conducted on liposomes (ligands bind onto the
outer membrane surfaces of liposomes) as DDS materials for use in
selective delivery of drugs or genes to a desired site, such as a cancer.
However, most of these liposomes bind to target cells ex vivo, but are
not targeted in vivo to expected target cells or tissues (see Forssen, E.
and Willis, M. (1998) Adv. Drug Delivery Rev. 29, 249-271; and Edited by
Toshio Takahashi-Mitsuru Hashida (1999), Today's DDS-drug delivery
system, pp. 159-167, Iyaku (Medicine and Drug) Journal Co., Ltd., Osaka,
Japan)). Also, in research and development concerning DDS materials using
the molecular recognition functions of sugar chains, some studies
concerning liposomes in which glycolipids having sugar chains have been
introduced are known. The functional evaluation of these liposomes has
been made ex vivo (in vitro) alone. Studies concerning liposomes into
which glycoproteins having sugar chains are introduced have remained
almost entirely unadvanced (see DeFrees, S. A., Phillips, L., Guo, L. and
Zalipsky, S. (1996) J. Am. Chem. Soc. 118, 6101-6104; Spevak, W., Foxall,
C., Charych, D. H., Dasqupta, F. and Nagy, J. O. (1996) J. Med. Chem. 39,
1018-1020; Stahn, R., Schafer, H., Kernchen, F. and Schreiber, J. (1998)
Glycobiology 8, 311-319; and Yamazaki, N., Jigami, Y., Gabius, H.-J.,
Kojima, S. (2001) Trends in Glycoscience and Glycotechnology 13, 319-329.
http://www.gak.co.jp/TIGG/71PDF/yamazaki.pdf). Therefore, systematic
studies including methods for preparing liposomes to which a wide variety
of sugar chains of glycolipids or glycoproteins are bound and in vivo
kinetics (in vivo) analysis are important issues that have not been
developed and are expected to be advanced in the future. As a study
concerning a further new type of DDS material, development of a DDS
material that can be used via oral administration, by which
administration can be performed most conveniently at low cost, is also an
important issue. For example, a peptidic pharmaceutical product is
characterized by being generally water soluble, having a high molecular
weight, and having low mucosal permeability in the alimentary canal
(small intestine). Hence, such a product is digested by an enzyme, or the
like, so that the product is almost never intestinally absorbed, even
when it is administered orally. A study concerning ligand-bound liposomes
is thus attracting attention, regarding a DDS material for delivery of
high-molecular-weight pharmaceutical products, genes, or the like into
blood from the intestinal tract (see Lehr, C.-M. (2000) J. Controlled
Release 65, 19-29).

[0007]JP Patent Publication (Kohyo) No. 5-507519 A (1993) discloses a
pharmaceutical composition having a pharmaceutically acceptable carrier
and a compound that contains an ingredient selectively binding to a
selectin receptor. However, in this pharmaceutical composition, a sugar
chain is used for the purpose of oral administration as a remedy itself
for inhibiting inflammatory disease and other diseases mediated by cell
adhesion. Thus, the pharmaceutical composition is different from a
sugar-chain-modified liposome.

[0008]JP Patent Publication (Kohyo) No. 2004-517835 A discloses a
pharmaceutical composition for parenteral administration, which comprises
a liposome comprising a non-charged vesicle-forming lipid that contains a
polyethylene glycol derivatization amphiphilic vesicle-forming lipid and
a negatively charged vesicle-forming lipid. However, this pharmaceutical
composition is used for parenteral administration and thus is different
from the sugar-chain-modified liposome of the present invention, which is
particularly suitable for oral administration. This patent publication
has no descriptions concerning sugar chains.

[0009]The present inventors have developed a sugar-chain-modified liposome
to which a sugar chain is bound via a linker protein (JP Patent
Publication (Kokai) No. 2003-226638 A). Moreover, the present inventors
have also discovered that the type and binding amount of a sugar chain
may be involved in tropism for each target cell or target tissue (JP
Patent Publication (Kokai) No. 2003-226647 A; International Publication
No. 2005/011632 Pamphlet; International Publication No. 2005/011633
Pamphlet; and Noboru Yamazaki (2005), Development of Active Targeting DDS
Nanoparticle, Bulletin of the Society of Nano Science and Technology, 3,
97-102). However, to date, no optimum sugar-chain-modified liposome that
can be used in various applications has been developed. Moreover, no
systematic studies have been conducted concerning sugar chains useful for
administration via various routes. It remains unknown about specifically
what kind of sugar chain should be used.

[0010]Therefore, there is a demand for a method for designing an optimum
delivery preparation with the use of a convenient experiment and assay.

DISCLOSURE OF THE INVENTION

Objects to be Achieved by the Invention

[0011]The objects of the present invention are to provide a method for
designing an optimum delivery preparation with the use of a convenient
experiment and/or assay and to search for the thus produced delivery
vehicle.

Means to Achieve the Objects

[0012]As a result of intensive studies to achieve the above objects, the
present inventors have discovered that a delivery vehicle (hereinafter,
also referred to as rolling model) that is useful in vivo can be produced
with a constant probability based on results obtained by processing
results obtained with the use of an in vitro assay system via specific
information processing. Hence, the present inventors have achieved the
above objects.

[0013]Therefore, the present invention provides the following (1) to
(111).

(1) A delivery vehicle for achieving delivery to a desired site, in which
binding takes place based on strong binding or weak binding to a cell
surface molecule associated with a desired site.(2) A delivery vehicle
for achieving delivery to a desired site, in which binding to a cell
surface molecule associated with a desired site is based on a rolling
model.(3) A delivery vehicle for achieving delivery to a desired site, in
which a strong binding inhibitory concentration (IC) involved in in vitro
affinity is low for a cell surface molecule associated with a desired
site.(4) A delivery vehicle for achieving delivery to a desired site, in
which a weak binding IC involved in in vitro affinity is high for a cell
surface molecule associated with a desired site.(5) A delivery vehicle
for achieving delivery to a desired site, in which a strong binding IC
involved in in vitro affinity for a cell surface molecule associated with
the desired site is low and a weak binding IC involved in in vitro
affinity for the same is high.(6) A delivery vehicle for achieving
delivery to a desired site, which contains at least one binding type from
among binding to a cell surface molecule associated with the desired site
based on a rolling model and binding based on other forms of strong
binding or weak binding.(7) The delivery vehicle according to any one of
(1) to (6), in which the cell surface molecule is selected from the group
consisting of a lectin, an adhesion molecule, an integrin, an
immunoglobulin, a sialomucin, a cadherin, a protein, a lipid, a receptor,
an antigen, an enzyme, a metalloprotease, a tyrosine phosphatase, a
glycolipid, a glycoprotein, a proteoglycan, a costimuratory molecule, a
membrane protein, and an extracellular matrix.(8) The delivery vehicle
according to any one of (1) to (6), in which the cell surface molecule is
a lectin.(9) The delivery vehicle according to any one of (1) to (6), in
which the boundary between the strong binding IC and the weak binding IC
is between 30 and 31 for "n" of ICn.(10) The delivery vehicle according
to any one of (1) to (6), in which when the strong binding IC is measured
under a condition such that "n" of ICn is 30 or less and the weak binding
IC is measured under a condition such that "n" of ICn is 31 or more.(11)
The delivery vehicle according to any one of (1) to (6), in which the
boundary between the strong binding IC and the weak binding IC is between
30 and 31 for "n" of ICn.(12) The delivery vehicle according to (3), in
which an inhibitory concentration at a strong IC which is IC30 or less is
10-9M or less as to in vitro affinity for a lectin associated with a
desired site.(13) The delivery vehicle according to (4), in which an
inhibitory concentration at a weak binding IC which is approximately IC31
or more is 10-9M or more as to in vitro affinity for a lectin
associated with a desired site.(14) The delivery vehicle according to
(5), in which an inhibitory concentration at a strong binding IC which is
approximately IC30 or less is 10-9M or less and an inhibitory
concentration at a weak binding IC which is approximately IC31 or more is
10-9M or more as to in vitro affinity for a lectin associated with a
desired site.(15) The delivery vehicle according to (14), which satisfies
at least one condition selected from the group consisting of a condition
that the inhibitory concentration at IC10 is 10-9M or less, a
condition that an inhibitory concentration at IC20 is 10-9M or less
and a condition that an inhibitory concentration at IC30 is 10-9M or
less for the strong binding IC as to in vitro affinity for a lectin
associated with a desired site, and satisfies at least one condition
selected from the group consisting of a condition that the inhibitory
concentration at IC40 is 10-9M or more, a condition that the
inhibitory concentration at IC50 is 10-9M or more and a condition
that the inhibitory concentration at IC60 is 10-9M or more for the
weak binding IC as to in vitro affinity for a lectin associated with a
desired site.(16) The delivery vehicle according to (15), in which at
least either the strong binding IC or the weak binding IC satisfies at
least two of the above conditions.(17) The delivery vehicle according to
any one of (1) to (16), in which the delivery vehicle is a liposome.(18)
The delivery vehicle according to any one of (1) to (16), in which the
delivery vehicle is a sugar-chain-modified liposome.(19) The delivery
vehicle according to any one of (1) to (16), in which the IC is measured
based on affinity for E-selectin.(20) The delivery vehicle according to
any one of (1) to (16), in which the desired site is selected from the
group consisting of an inflammation site and a cancer site.(21) The
delivery vehicle according to (12), which contains a liposome selected
from the group consisting of liposome No. 16, liposome No. 27, liposome
No. 29, liposome No. 41, liposome No. 45, liposome No. 53, liposome No.
69, liposome No. 71, liposome No. 76, liposome No. 80, liposome No. 87,
liposome No. 91, liposome No. 93, liposome No. 96, liposome No. 105,
liposome No. 106, liposome No. 117, liposome No. 125, liposome No. 127,
liposome No. 137, liposome No. 139, liposome No. 142, liposome No. 146,
liposome No. 150, liposome No. 151, liposome No. 152, liposome No. 153,
liposome No. 154, liposome No. 184, liposome No. 186, liposome No. 191,
liposome No. 195, liposome No. 199, liposome No. 204, liposome No. 207,
liposome No. 213, liposome No. 224, liposome No. 225, liposome No. 229,
liposome No. 230, liposome No. 234, liposome No. 235, liposome No. 239,
liposome No. 240, liposome No. 263, liposome No. 273, liposome No. 285,
and liposome No. 295.(22) The delivery vehicle according to (13), which
contains a liposome selected from the group consisting of liposome No. 3,
liposome No. 16, liposome No. 27, liposome No. 29, liposome No. 38,
liposome No. 40, liposome No. 41, liposome No. 45, liposome No. 50,
liposome No. 53, liposome No. 56, liposome No. 60, liposome No. 68,
liposome No. 69, liposome No. 70, liposome No. 71, liposome No. 76,
liposome No. 80, liposome No. 87, liposome No. 91, liposome No. 93,
liposome No. 96, liposome No. 105, liposome No. 106, liposome No. 111,
liposome No. 116, liposome No. 117, liposome No. 120, liposome No. 125,
liposome No. 127, liposome No. 129, liposome No. 130, liposome No. 137,
liposome No. 139, liposome No. 141, liposome No. 142, liposome No. 146,
liposome No. 150, liposome No. 151, liposome No. 152, liposome No. 153,
liposome No. 154, liposome No. 155, liposome No. 175, liposome No. 178,
liposome No. 183, liposome No. 184, liposome No. 186, liposome No. 191,
liposome No. 195, liposome No. 197, liposome No. 199, liposome No. 204,
liposome No. 207, liposome No. 209, liposome No. 213, liposome No. 218,
liposome No. 220, liposome No. 224, liposome No. 225, liposome No. 229,
liposome No. 230, liposome No. 233, liposome No. 234, liposome No. 235,
liposome No. 236, liposome No. 237, liposome No. 239, liposome No. 240,
liposome No. 254, liposome No. 263, liposome No. 273, liposome No. 285,
liposome No. 290, liposome No. 292, and liposome No. 295.(23) The
delivery vehicle according to (14), which contains a liposome selected
from the group consisting of liposome No. 16, liposome No. 27, liposome
No. 29, liposome No. 41, liposome No. 45, liposome No. 53, liposome No.
69, liposome No. 71, liposome No. 76, liposome No. 80, liposome No. 87,
liposome No. 91, liposome No. 93, liposome No. 96, liposome No. 105,
liposome No. 106, liposome No. 111, liposome No. 125, liposome No. 127,
liposome No. 137, liposome No. 139, liposome No. 142, liposome No. 146,
liposome No. 150, liposome No. 151, liposome No. 152, liposome No. 153,
liposome No. 154, liposome No. 184, liposome No. 186, liposome No. 191,
liposome No. 195, liposome No. 199, liposome No. 204, liposome No. 207,
liposome No. 213, liposome No. 224, liposome No. 225, liposome No. 229,
liposome No. 230, liposome No. 234, liposome No. 235, liposome No. 239,
liposome No. 240, liposome No. 263, liposome No. 273, liposome No. 285,
and liposome No. 295.(24) The delivery vehicle according to (15), which
contains a liposome selected from the group consisting of liposome No. 3,
liposome No. 16, liposome No. 27, liposome No. 29, liposome No. 38,
liposome No. 40, liposome No. 41, liposome No. 45, liposome No. 50,
liposome No. 53, liposome No. 56, liposome No. 60, liposome No. 68,
liposome No. 69, liposome No. 70, liposome No. 71, liposome No. 76,
liposome No. 80, liposome No. 87, liposome No. 91, liposome No. 93,
liposome No. 96, liposome No. 105, liposome No. 106, liposome No. 111,
liposome No. 116, liposome No. 117, liposome No. 120, liposome No. 125,
liposome No. 127, liposome No. 129, liposome No. 130, liposome No. 137,
liposome No. 139, liposome No. 141, liposome No. 142, liposome No. 146,
liposome No. 150, liposome No. 151, liposome No. 152, liposome No. 153,
liposome No. 154, liposome No. 155, liposome No. 175, liposome No. 178,
liposome No. 183, liposome No. 184, liposome No. 186, liposome No. 191,
liposome No. 195, liposome No. 197, liposome No. 199, liposome No. 204,
liposome No. 207, liposome No. 209, liposome No. 213, liposome No. 218,
liposome No. 220, liposome No. 224, liposome No. 225, liposome No. 229,
liposome No. 230, liposome No. 233, liposome No. 234, liposome No. 235,
liposome No. 236, liposome No. 237, liposome No. 239, liposome No. 240,
liposome No. 254, liposome No. 263, liposome No. 273, liposome No. 285,
liposome No. 290, liposome No. 292, and liposome No. 295.(25) The
delivery vehicle according to (16), which contains a liposome selected
from the group consisting of liposome No. 3, liposome No. 16, liposome
No. 27, liposome No. 29, liposome No. 38, liposome No. 40, liposome No.
41, liposome No. 45, liposome No. 50, liposome No. 53, liposome No. 56,
liposome No. 60, liposome No. 68, liposome No. 69, liposome No. 70,
liposome No. 71, liposome No. 76, liposome No. 80, liposome No. 87,
liposome No. 91, liposome No. 93, liposome No. 96, liposome No. 105,
liposome No. 106, liposome No. 111, liposome No. 116, liposome No. 117,
liposome No. 120, liposome No. 125, liposome No. 127, liposome No. 129,
liposome No. 130, liposome No. 137, liposome No. 139, liposome No. 141,
liposome No. 142, liposome No. 146, liposome No. 150, liposome No. 151,
liposome No. 152, liposome No. 153, liposome No. 154, liposome No. 155,
liposome No. 175, liposome No. 178, liposome No. 183, liposome No. 184,
liposome No. 186, liposome No. 191, liposome No. 195, liposome No. 197,
liposome No. 199, liposome No. 204, liposome No. 207, liposome No. 209,
liposome No. 213, liposome No. 218, liposome No. 220, liposome No. 224,
liposome No. 225, liposome No. 229, liposome No. 230, liposome No. 233,
liposome No. 234, liposome No. 235, liposome No. 236, liposome No. 237,
liposome No. 239, liposome No. 240, liposome No. 254, liposome No. 263,
liposome No. 273, liposome No. 285, liposome No. 290, liposome No. 292,
and liposome No. 295.(26) A method for producing a delivery vehicle for
achieving the delivery of a desired substance to a desired site, which
comprises the steps of:A) measuring in vitro affinity of candidate
delivery vehicles for a cell surface molecule associated with the site;
andB) selecting a delivery vehicle having in vitro affinity corresponding
to delivery to the desired site.(27) The method according to (26), in
which the delivery vehicle contains a liposome.(28) The method according
to (26), in which the delivery vehicle contains a sugar-chain-modified
liposome.(29) The method according to (26), in which the candidates
contain sugar-chain-modified liposomes containing a plurality of sugar
chain type.(30) The method according to (26), in which the cell surface
molecule is selected from the group consisting of a lectin, an adhesion
molecule, an integrin, an immunoglobulin, a sialomucin, a cadherin, a
protein, a lipid, a receptor, an antigen, an enzyme, a metalloprotease, a
tyrosine phosphatase, a glycolipid, a glycoprotein, a proteoglycan, a
costimuratory molecule, a membrane protein, and an extracellular
matrix.(31) The method according to (26), in which the cell surface
molecule is a lectin.(32) The method according to (26), in which the cell
surface molecule contains a lectin selected from the group consisting of
E-selectin, P-selectin, L-selectin, galectin 1, galectin 2, galectin 3,
galectin 4, galectin 5, galectin 6, galectin 7, galectin 8, galectin 9,
galectin 10, galectin 11, galectin 12, galectin 13, galectin 14,
mannose-6-phosphate receptor, calnexin, calreticulin, ERGIC-53, VIP53,
interleukins, interferons, and growth factors.(33) The method according
to (26), in which the cell surface molecule contains E-selectin.(34) The
method according to (26), in which the cell surface molecule contains
E-selectin and the site is selected from the group consisting of oral
administration, a site of the liver, a site of the small intestine, a
site of the large intestine, a site of the lymph node, a site of the
heart, a site of the pancreas, a site of the lungs, a site of the brain,
a site of the bone marrow, a site in blood, a site of the kidney, a site
of the spleen, a site of the thymus gland, a site of muscle, an
inflammation site, and a cancer site.(35) The method according to (26),
in which the cell surface molecule contains E-selectin and the site is
selected from the group consisting of a tumor site and an inflammation
site.(36) The method according to (26), in which the affinity is
represented by n % inhibitory concentration (ICn), wherein "n" ranges
from 1 to 99.(37) The method according to (26), in which the measurement
of affinity comprises measurement at a strong binding IC that is
approximately IC30 or less and measurement at a weak binding IC that is
approximately IC31 or more.(38) The method according to (26), in which
the measurement of affinity comprises measurement at a strong binding IC
that is at least one point between IC30 and IC10 and measurement at a
weak binding IC that is at least one point between IC40 and IC60.(39) The
method according to (26), in which the measurement of affinity comprises
measurement at a strong binding IC that is approximately IC30 or less and
a candidate having a low inhibitory concentration at the strong binding
IC is selected.(40) The method according to (26), in which the
measurement of affinity comprises measurement at a strong binding IC that
is approximately IC30 or less and a candidate having an inhibitory
concentration of 10-9M or less at the strong binding IC is
selected.(41) The method according to (26), in which the measurement of
affinity comprises measurement at a strong binding IC that is
approximately IC30 or less and a candidate having a low inhibitory
concentration at the strong binding IC is selected, whereinthe selection
is performed when at least one condition selected from the group
consisting of a condition that the inhibitory concentration at IC10 is 10

-9M or less, a condition that an inhibitory concentration at IC20 is
10-9M or less and a condition that an inhibitory concentration at
IC30 is 10-9M or less is satisfied.(42) The method according to
(26), in which the measurement of affinity comprises measurement at a
weak binding IC that is approximately IC31 or more and a candidate having
a high inhibitory concentration at the strong binding IC is selected.(43)
The method according to (26), in which the measurement of affinity
comprises measurement at a weak binding IC that is approximately IC31 or
more and a candidate having an inhibitory concentration of 10-9M or
more at the weak binding IC is selected.(44) The method according to
(26), in which the measurement of affinity comprises measurement at a
weak binding IC that is approximately IC31 or more and a candidate having
a high inhibitory concentration at the weak binding IC is selected,
whereinthe selection is performed when at least one condition selected
from the group consisting of a condition that the inhibitory
concentration at IC40 is 10-9M or more, a condition that the
inhibitory concentration at IC50 is 10-9M or more and a condition
that the inhibitory concentration at IC60 is 10-9M or more is
satisfied.(45) The method according to (26), in which the measurement of
affinity comprises measurement at a strong binding IC that is
approximately IC30 or less and a weak binding IC that is approximately
IC31 or more and a candidate having a low inhibitory concentration at the
strong binding IC and a high inhibitory concentration at the weak binding
IC is selected.(46) The method according to (26), in which the
measurement of affinity comprises measurement at a strong binding IC that
is approximately IC30 or less and a weak binding IC that is approximately
IC31 or more and a candidate having an inhibitory concentration of
10-9M or less at the strong binding IC and an inhibitory
concentration of 10-9M or more at the weak binding IC is
selected.(47) The method according to (26), in which the measurement of
affinity comprises measurement at a strong binding IC that is
approximately IC30 or less and a weak binding IC that is approximately
IC31 or more and a candidate having a low inhibitory concentration at the
strong binding IC and a high inhibitory concentration at the weak binding
IC is selected,whereinthe selection is performed when at least one
condition selected from the group consisting of a condition that the
inhibitory concentration at IC10 is 10-9M or less, a condition that
an inhibitory concentration at IC20 is 10-9M or less and a condition
that an inhibitory concentration at IC30 is 10-9M or less is
satisfied; and in terms of the weak binding IC, at least one condition
selected from the group consisting of a condition that the inhibitory
concentration at IC40 is 10-9M or more, a condition that the
inhibitory concentration at IC50 is 10-9M or more and a condition
that the inhibitory concentration at IC60 is 10-9M or more is
satisfied.(48) The method according to (26), in which the measurement of
affinity is performed by a method selected from the group consisting of a
competitive inhibition assay, a noncompetitive inhibition assay, and a
binding assay.(49) A method for producing a delivery vehicle for
achieving delivery to a desired site, which comprises the steps of:A)
measuring in vitro affinity of candidate delivery vehicles for a cell
surface molecule associated with the site; andB) selecting a delivery
vehicle having in vitro affinity corresponding to delivery to the desired
site and analyzing the composition of the selected delivery vehicle;
andC) generating the thus selected delivery vehicle based on the
composition.(50) The method according to (49), which further comprises
the step of causing the selected delivery vehicle to contain a substance
to be delivered.(51) The method according to (49), in which the delivery
vehicle contains a liposome.(52) The method according to (49), in which
the delivery vehicle contains a sugar-chain-modified liposome.(53) The
method according to (49), in which the candidate contains a
sugar-chain-modified liposome that contains a plurality of sugar chain
types.(54) The method according to (49), in which the cell surface
molecule is selected from the group consisting of a lectin, an adhesion
molecule, an integrin, an immunoglobulin, a sialomucin, a cadherin, a
protein, a lipid, a receptor, an antigen, an enzyme, a metalloprotease, a
tyrosine phosphatase, a glycolipid, a glycoprotein, a proteoglycan, a
costimuratory molecule, a membrane protein, and an extracellular
matrix.(55) The method according to (49), in which the cell surface
molecule is a lectin.(56) The method according to (49), in which the cell
surface molecule contains a lectin selected from the group consisting of
E-selectin, P-selectin, L-selectin, galectin 1, galectin 2, galectin 3,
galectin 4, galectin 5, galectin 6, galectin 7, galectin 8, galectin 9,
galectin 10, galectin 11, galectin 12, galectin 13, galectin 14, a
mannose-6-phosphate receptor, calnexin, calreticulin, ERGIC-53, VIP53,
interleukins, interferons, and growth factors.(57) The method according
to (49), in which the cell surface molecule contains E-selectin.(58) The
method according to (49), in which the cell surface molecule contains
E-selectin and the site is selected from the group consisting of oral
administration, a site of the liver, a site of the small intestine, a
site of the large intestine, a site of the lymph node, a site of the
liver, a site of the heart, a site of the pancreas, a site of the lungs,
a site of the brain, a site of the bone marrow, a site in blood, a site
of the kidney, a site of the spleen, a site of the thymus gland, a site
of muscle, an inflammation site, and a cancer site.(59) The method
according to (49), in which the cell surface molecule contains E-selectin
and the site is selected from the group consisting of a tumor site and an
inflammation site.(60) The method according to (49), in which the
affinity is represented by ICn, wherein "n" ranges from 1 to 99.(61) The
method according to (49), in which the measurement of affinity comprises
measurement at a strong binding IC that is approximately IC30 or less and
measurement at a weak binding IC that is approximately IC31 or more.(62)
The method according to (49), in which the measurement of affinity
comprises measurement at a strong binding IC that is at least one between
IC30 and IC10 and measurement at a weak binding IC that is at least one
between IC40 and IC60.(63) The method according to (49), in which the
measurement of affinity comprises measurement at a strong binding IC that
is approximately IC30 or less and a candidate having a low inhibitory
concentration at the strong binding IC is selected.(64) The method
according to (49), in which the measurement of affinity comprises
measurement at a strong binding IC that is approximately IC30 or less and
a candidate having an inhibitory concentration of 10-9M or less at
the strong binding IC is selected.(65) The method according to (49), in
which the measurement of affinity comprises measurement at a strong
binding IC that is approximately IC30 or less and a candidate having a
low inhibitory concentration at the strong binding IC is selected,
whereinthe selection is performed when at least one condition selected
from the group consisting of a condition that the inhibitory
concentration at IC10 is 10-9M or less, a condition that an
inhibitory concentration at IC20 is 10-9M or less and a condition
that an inhibitory concentration at IC30 is 10-9M or less is
satisfied.(66) The method according to (49), in which the measurement of
affinity comprises measurement at a weak binding IC that is approximately
IC31 or more and a candidate having a high inhibitory concentration at
the strong binding IC is selected.(67) The method according to (49), in
which the measurement of affinity comprises measurement at a weak binding
IC that is approximately IC31 or more and a candidate having an
inhibitory concentration of 10-9M or more at the weak binding IC is
selected.(68) The method according to (49), in which the measurement of
affinity comprises measurement at a weak binding IC that is approximately
IC31 or more and a candidate having a high inhibitory concentration at
the weak binding IC is selected, whereinthe selection is performed when
at least one condition selected from the group consisting of a condition
that the inhibitory concentration at IC60 is 10-9M or more, a
condition that the inhibitory concentration at IC50 is 10-9M or more
and a condition that the inhibitory concentration at IC40 is 10-9M
or more is satisfied.(69) The method according to (49), in which the
measurement of affinity comprises measurement at a strong binding IC that
is approximately IC30 or less and a weak binding IC that is approximately
IC31 or more and a candidate having a low inhibitory concentration at the
strong binding IC and a high inhibitory concentratin at the weak binding
IC is selected.(70) The method according to (49), in which the
measurement of affinity comprises measurement at a strong binding IC that
is approximately IC30 or less and a weak binding IC that is approximately
IC31 or more and a candidate having an inhibitory concentration of
10-9M or less at the strong binding IC and an inhibitory
concentration of 10-9M or more at the weak binding IC is
selected.(71) The method according to (49), in which the measurement of
affinity comprises measurement at a strong binding IC that is
approximately IC30 or less and a weak binding IC that is approximately
IC31 or more and a candidate having a low inhibitory concentration at the
strong binding IC and a high inhibitory concentration at the weak binding
IC is selected,whereinthe selection is performed when at least one
condition selected from the group consisting of a condition that the
inhibitory concentration at IC10 is 10-9M or less, a condition that
an inhibitory concentration at IC20 is 10-9M or less and a condition
that an inhibitory concentration at IC30 is 10-9M or less is
satisfied; and in terms of the weak binding IC, at least one condition
selected from the group consisting of a condition that the inhibitory
concentration at IC40 is 10-9M or more, a condition that the
inhibitory concentration at IC50 is 10-9M or more and a condition
that the inhibitory concentration at IC60 is 10-9M or more is
satisfied.(72) The method according to (49), in which the measurement of
affinity is performed by a method selected from the group consisting of a
competitive inhibition assay, a noncompetitive inhibition assay, and a
binding assay.(73) The method according to (49), which further comprises
a step of determining a method for preparing a delivery vehicle having
the composition in addition to the analysis of the composition.(74) The
method according to (49), in which the delivery vehicle contains a
sugar-chain-modified liposome and the analysis of the composition
comprises analysis of the sugar chain type and density of the
sugar-chain-modified liposome.(75) The method according to (49), in which
the delivery vehicle contains a sugar-chain-modified liposome and the
analysis of the composition comprises determination of a method for
producing the sugar-chain-modified liposome.(76) The method according to
(49), in which the delivery vehicle contains a sugar-chain-modified
liposome and which comprises determination of a method for producing the
sugar-chain-modified liposome instead of the analysis of the
composition.(77) The method according to (49), in which the delivery
vehicle contains a sugar-chain-modified liposome and which further
comprises, when the sugar-chain-modified liposome is generated, a step of
causing a reaction of a sugar chain, the type and the extent of which is
determined based on the above composition, under appropriate conditions
for binding to the liposome.(78) The method according to (57), in which a
linker is used in the binding.(79) The method according to (78), in which
the linker is a protein.(80) The method according to (78), in which the
linker is an albumin.(81) The method according to (77), which further
comprises a step of hydrophilizing the liposome.(82) The method according
to (49), which further comprises a step of confirming the in vivo dynamic
state of the selected delivery vehicle.(83) A method for producing a
delivery vehicle by which delivery to an undesired site does not occur,
which comprises the steps of:A) measuring in vitro affinity of candidate
delivery vehicles for a cell surface molecule associated with the site;
andB) selecting a delivery vehicle having in vitro affinity corresponding
to non-delivery to the undesired site.(84) The method according to (83),
in which the cell surface molecule is selected from the group consisting
of a lectin, an adhesion molecule, an integrin, an immunoglobulin, a
sialomucin, a cadherin, a protein, a lipid, a receptor, an antigen, an
enzyme, a metalloprotease, a tyrosine phosphatase, a glycolipid, a
glycoprotein, a proteoglycan, a costimuratory molecule, a membrane
protein, and an extracellular matrix.(85) The method according to (83),
in which the cell surface molecule is a lectin.(86) A method for
producing a delivery vehicle by which delivery to an undesired site does
not occur, which comprises the steps of:A) measuring in vitro affinity of
candidate delivery vehicles for a cell surface molecule associated with
the site;B) selecting a delivery vehicle having in vitro affinity
corresponding to non-delivery to the undesired site and analyzing the
composition of the selected delivery vehicle; andC) generating the
selected delivery vehicle based on the composition.(87) The method
according to (86), in which the cell surface molecule is selected from
the group consisting of a lectin, an adhesion molecule, an integrin, an
immunoglobulin, a sialomucin, a cadherin, a protein, a lipid, a receptor,
an antigen, an enzyme, a metalloprotease, a tyrosine phosphatase, a
glycolipid, a glycoprotein, a proteoglycan, a costimuratory molecule, a
membrane protein, and an extracellular matrix.(88) The method according
to (86), in which the cell surface molecule is a lectin.(89) A method for
producing a delivery vehicle for achieving specific delivery, which
comprises the steps of:A) measuring in vitro affinity of candidate
delivery vehicles for a cell surface molecule associated with the site to
which specific delivery is performed;B) measuring in vitro affinity of
the candidate delivery vehicles for a cell surface molecule associated
with a site to which specific delivery is not performed; andC) selecting
a delivery vehicle having in vitro affinity corresponding to delivery to
a desired site and corresponding to non-delivery to an undesired
site.(90) The method according to (89), in which the cell surface
molecule is selected from the group consisting of a lectin, an adhesion
molecule, an integrin, an immunoglobulin, a sialomucin, a cadherin, a
protein, a lipid, a receptor, an antigen, an enzyme, a metalloprotease, a
tyrosine phosphatase, a glycolipid, a glycoprotein, a proteoglycan, a
costimuratory molecule, a membrane protein, and an extracellular
matrix.(91) The method according to (89), in which the cell surface
molecule is a lectin.(92) A method for producing a delivery vehicle for
achieving specific delivery, which comprises the steps of:A) measuring in
vitro affinity of candidate delivery vehicles for a cell surface molecule
associated with the site to which specific delivery is performed;B)
measuring in vitro affinity of the candidate delivery vehicles for a cell
surface molecule associated with a site to which specific delivery is not
performed;C) selecting a delivery vehicle having in vitro affinity
corresponding to delivery to a desired site and corresponding to
non-delivery to an undesired site and analyzing the composition of the
selected delivery vehicle; andD) generating the selected delivery vehicle
based on the composition.(93) The method according to (92), in which the
cell surface molecule is selected from the group consisting of a lectin,
an adhesion molecule, an integrin, an immunoglobulin, a sialomucin, a
cadherin, a protein, a lipid, a receptor, an antigen, an enzyme, a
metalloprotease, a tyrosine phosphatase, a glycolipid, a glycoprotein, a
proteoglycan, a costimuratory molecule, a membrane protein, and an
extracellular matrix.(94) The method according to (92), in which the cell
surface molecule is a lectin.(95) A delivery vehicle, which is produced
by the method according to any one of (26) to (94).(96) A method for
preventing or treating a subject who requires delivery of a drug to a
desired site, which comprises the steps of:A) measuring in vitro affinity
of candidate delivery vehicles, which are intended for achieving delivery
to a desired site, for a cell surface molecule associated with the
desired site;B) selecting a delivery vehicle having in vitro affinity
corresponding to delivery to the desired site; andC) administering a drug
required for prevention or treatment to the subject with the use of the
selected delivery vehicle.(97) The method according to (96), in which the
cell surface molecule is a lectin, an adhesion molecule, an integrin, an
immunoglobulin, a sialomucin, a cadherin, a protein, a lipid, a receptor,
an antigen, an enzyme, a metalloprotease, a tyrosine phosphatase, a
glycolipid, a glycoprotein, a proteoglycan, a costimuratory molecule, a

membrane protein, and an extracellular matrix.(98) The method according
to (96), in which the cell surface molecule is a lectin.(99) A method for
preventing or treating a subject who requires delivery of a drug to a
desired site, which comprises the steps of:A) measuring in vitro affinity
of candidate delivery vehicles, which are intended for achieving delivery
to a desired site, for a cell surface molecule associated with the
site;B) selecting a delivery vehicle having in vitro affinity
corresponding to delivery to the desired site and analyzing the
composition of the selected delivery vehicle;C) generating the selected
delivery vehicle containing a drug required for prevention or treatment
based on the composition; andD) administering the selected delivery
vehicle to the subject.(100) The method according to (99), in which the
cell surface molecule is selected from the group consisting of a lectin,
an adhesion molecule, an integrin, an immunoglobulin, a sialomucin, a
cadherin, a protein, a lipid, a receptor, an antigen, an enzyme, a
metalloprotease, a tyrosine phosphatase, a glycolipid, a glycoprotein, a
proteoglycan, a costimuratory molecule, a membrane protein, and an
extracellular matrix.(101) The method according to (99), in which the
cell surface molecule is a lectin.(102) An apparatus for producing a
delivery vehicle for achieving delivery to a desired site, which is
provided with:A) a means for measuring in vitro affinity of candidate
delivery vehicles for a cell surface molecule associated with the site;
andB) a means for selecting a delivery vehicle having in vitro affinity
corresponding to delivery to the desired site.(103) The apparatus
according to (102), in which the cell surface molecule is selected from
the group consisting of a lectin, an adhesion molecule, an integrin, an
immunoglobulin, a sialomucin, a cadherin, a protein, a lipid, a receptor,
an antigen, an enzyme, a metalloprotease, a tyrosine phosphatase, a
glycolipid, a glycoprotein, a proteoglycan, a costimuratory molecule, a
membrane protein, and an extracellular matrix.(104) The apparatus
according to (102), in which the cell surface molecule is a lectin.(105)
An apparatus for producing a delivery vehicle for achieving delivery to a
desired site, which is provided with:A) a means for measuring in vitro
affinity of candidate delivery vehicles for a cell surface molecule
associated with the site;B) a means for selecting a delivery vehicle
having in vitro affinity corresponding to delivery to the desired site.C)
a means for analyzing the composition of the selected delivery vehicle;
andD) a means for generating the selected delivery vehicle based on the
composition.(106) The apparatus according to (105), in which the cell
surface molecule is selected from the group consisting of a lectin, an
adhesion molecule, an integrin, an immunoglobulin, a sialomucin, a
cadherin, a protein, a lipid, a receptor, an antigen, an enzyme, a
metalloprotease, a tyrosine phosphatase, a glycolipid, a glycoprotein, a
proteoglycan, a costimuratory molecule, a membrane protein, and an
extracellular matrix.(107) The apparatus according to (105), in which the
cell surface molecule is a lectin.(108) Use of in vitro affinity for a
cell surface molecule associated with a desired site, which is intended
for producing a delivery vehicle for achieving delivery to the desired
site.(109) The use according to (108), in which the cell surface molecule
is selected from the group consisting of a lectin, an adhesion molecule,
an integrin, an immunoglobulin, a sialomucin, a cadherin, a protein, a
lipid, a receptor, an antigen, an enzyme, a metalloprotease, a tyrosine
phosphatase, a glycolipid, a glycoprotein, a proteoglycan, a
costimuratory molecule, a membrane protein, and an extracellular
matrix.(110) The use according to (108), in which the cell surface
molecule is a lectin.(111) A pharmaceutical composition, which contains a
drug to be used for prevention or treatment, and the delivery vehicle
according to any one of (1) to (25) or a delivery vehicle that is
produced by the method according to any one of (26) to (94).

[0014]Hereafter, preferred embodiments of the present invention will be
described. It should be recognized that persons skilled in the art can
adequately implement the embodiments according to the explanation and
attached drawings of the present invention and known conventional
technology in the art and can easily understand the action and effect
exerted by the present invention.

EFFECT OF THE INVENTION

[0015]According to the present invention, a method for designing a useful
delivery vehicle such as a sugar-chain-modified liposome, a production
method based on a rolling model, and a method for using them are
provided. The delivery vehicle of the present invention significantly
increases the range of development of a DDS formulation with which a
desired drug can be provided to a target delivery site. The present
invention enables development and practical application of a delivery
system that is required for realization of new treatment in each field of
cancer therapy, gene therapy, regeneration medicine, and the like. Such
various delivery vehicles that are useful for oral administration are
provided according to the present invention for the first time.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1 is a schematic diagram showing the production of a
sugar-chain-modified liposome that can be used in the present invention.

[0017]FIG. 2 is a graph for calculation of ICn. IC50 is shown in this
figure. The concentration of a subject drug is plotted on the X axis and
the amount of a bound ligand is plotted on the Y axis.

[0018]FIG. 3 is a graph for calculation of IC20. LNT-4, LY-1, and A6 are
abbreviated names of liposomes.

[0019]FIG. 4 is a graph for calculation of IC30. LNT-4, LY-1, and A6 are
abbreviated names of liposomes.

[0020]FIG. 5 is a graph for calculation of IC40. LNT-4, LY-1, and A6 are
abbreviated names of liposomes.

[0021]FIG. 6 is a graph for calculation of IC50. LNT-4, LY-1, and A6 are
abbreviated names of liposomes.

[0023]FIG. 8 shows fluorescence microscopic photographs showing the effect
of accumulating doxorubicin from tumor blood vessels to tumor tissues in
cancer-bearing mice to which doxorubicin-encapsulated liposome No. 155
was injected via tail vein. Images on the left and images on the right
are green and red fluorescence microscopic photographs of the same tumor
tissue.

[0025]FIG. 10 shows fluorescence microscopic photographs showing the
effect of accumulating doxorubicin-encapsulated liposome No. 237 from
tumor blood vessels to tumor tissue, which was administered via oral
administration to cancer-bearing mice. Images on the left and images on
the right are green and red fluorescence microscopic photographs of the
same tumor tissue.

[0026]FIG. 11 is a typical graph of a rolling model showing
concentration-inhibition % curves (substitution curves). A solid line is
an example of a curve more preferred in the rolling model than a dotted
line. A dotted line is an unpreferable example (e.g., non-specific
binding or antigen antibody reaction).

[0027]FIG. 12 is a schematic diagram of Example 1 of the action mechanism
of a delivery vehicle based on a rolling model.

[0028]FIG. 13 is a schematic diagram of Example 2 of the action mechanism
of a delivery vehicle based on a rolling model.

[0029]FIG. 14 is a schematic diagram of Example 3 of the action mechanism
of a delivery vehicle based on a rolling model.

[0030]FIG. 15 is a schematic diagram of Example 4 of the action mechanism
of a delivery vehicle based on a rolling model.

BEST MODE OF CARRYING OUT THE INVENTION

[0031]Hereinafter, the present invention will be explained by describing
the best mode thereof. Throughout the description, expressions in the
singular form should be understood as including the concept of the plural
form thereof, unless otherwise specified. Therefore, articles in the
singular form (e.g., in the case of English, "a," "an," and "the") should
be understood as including the concept of the plural form thereof, unless
otherwise specified. Furthermore, it should be understood that terms used
in the description have the same meanings as those that generally apply
in the above fields, unless specified. Therefore, unless otherwise
defined, all the technical terms and engineering and scientific terms
that are used in the description have meanings that are generally
understood by persons skilled in the fields to which the present
invention corresponds. When there is a conflict, the description
(including definitions) is prioritized.

[0032]Embodiments that are provided hereafter are provided for better
understanding of the present invention. It is understood that the scope
of the present invention should not be limited to the following
description. Therefore, it is clear that persons skilled in the art can
adequately make modifications within the scope of the present invention
by taking the content of the description into consideration.

[0033]Preferred embodiments of the present invention are explained by
adequately explaining the definitions of terms that are particularly used
in the description. A sugar-chain-modified liposome is the main object of
explanation in the present invention, but it will be understood that the
rolling model of the present invention is not limited thereto.

(In Vitro Assay Using Cell Surface Molecule)

[0034]According to an aspect of the present invention, a method for
producing a delivery vehicle that is used for the achievement of delivery
of a desired substance to a desired site is provided. The production
method comprises the steps of: A) measuring in vitro affinity of delivery
vehicle candidates for a cell surface molecule such as a lectin
associated with the site; and B) selecting a delivery vehicle having in
vitro affinity corresponding to delivery to the desired site.

[0035]Alternatively, the production method of the present invention
comprises the steps of: A) measuring in vitro affinity of delivery
vehicle candidates for a cell surface molecule such as a lectin
associated with the site; B) selecting a delivery vehicle having in vitro
affinity corresponding to delivery to the desired site and analyzing the
composition of the selected delivery vehicle; and C) generating the
selected delivery vehicle based on the composition.

[0036]In the description, "delivery vehicle" refers to a vehicle that
mediates the delivery of a desired substance. If a substance to be
delivered is a drug, the delivery vehicle is referred to as a "drug
delivery vehicle." Examples of the delivery vehicle can include a lipid
vehicle, a liposome, a lipid micelle, a lipoprotein micelle, a
lipid-stabilizing emulsion, cyclodextrin, a polymer nanoparticle, a
polymer fine particle, a block copolymer micelle, a polymer-lipid hybrid
system, and a derivatized single-chain polymer.

[0037]DDS is also referred to as a drug delivery system and may be
classified into absorption-control DDS, release-control DDS, and
targeting DDS. An ideal DDS is a system which sends a drug "to sites
where it is needed in vivo," "in a required amount," and "only for
required time." Targeting DDS (Targeting DDS in English and "Hyo-teki
shiko-sei DDS" in Japanese) is classified into passive targeting DDS and
active targeting DDS. Passive targeting DDS uses the physicochemical
properties (e.g., particle diameter and hydrophilicity) of a carrier
(transporter of the drug) to control behavior in vivo. Active targeting
DDS adds special mechanisms to the passive type to actively control the
tropism for the target tissue. For example, there is a method referred to
as "missile drug" using carriers consisting of combinations of
antibodies, sugar chains, etc., that are capable of specifically
recognizing target molecules of certain cells that make up the target
tissue.

[0038]In the description, "encapsulation" means stable association with a
delivery vehicle. When a vehicle is administered in vivo, encapsulation
of one or a plurality of drugs is not required as long as one or a
plurality of drugs are stably associated with the vehicle. Therefore,
"stably associated with . . . " and "encapsulated in . . . " or
"encapsulated together with . . . " or "encapsulated in or encapsulated
together with . . . " are intended to be synonymous with each other.
These terms are used in the description interchangeably. Stable
association can be generated via various means including a covalent bond
with a delivery vehicle, preferably a bond that can be cleaved, a
non-covalent bond, capturing of a drug within a delivery vehicle, and the
like. Association must be sufficiently stable to such a degree that
association with a delivery vehicle is maintained at a noncompetitive
rate until the drug is delivered to a target site in a subject to which
the drug is administered.

[0039]It is understood that in the description, any substance can be used
as a delivery vehicle, as long as it is compatible with a living body (in
the description, referred to as "biocompatible substance") into which the
substance is delivered. Preferably, such a biocompatible substance can
preferably be a biodegradable substance (e.g., biodegradable polymer) and
may be any substance as long as it does not have any harmful effect on a
living body. Examples of such substance include a lipid (e.g., a
liposome), polyester, cyclodextrin, polyamino acid, silicon (e.g., porous
silicon or a biosilicon material (for example, substances disclosed in
WO99/53898, the disclosure of which is incorporated herein in its
entirety as a reference)), mesoporous, microporous, or polycrystalline
silicon), an ethylene vinyl acetate copolymer, and polyvinylalcohol.
Examples of a biodegradable polymer include, but are not limited to,
polyester (e.g., polylactic acid-glycolic acid copolymer (PLGA)),
hydrophobic polyamino acid (e.g., polyalanine and polyleucine),
polyanhydride, polyglycerol sebacate (PGS), and Biopol. "Hydrophobic
polyamino acid" means a polymer that is prepared from hydrophobic amino
acid. Examples of a non-biodegradable polymer that can be used in the
present invention include, but are not limited to, silicon,
polytetrafluoroethylene, polyethylene, polypropylene, polyurethane,
polyacrylate, polymethacrylate (e.g., polymethylmethacrylate), and
ethylene vinyl acetate copolymer.

[0040]The delivery vehicle of the present invention may also contain a
water-soluble substance. A water-soluble substance plays a role in
control of the water infiltration into a drug dispersion system. Such
water-soluble substances are not limited in view of water-soluble
substance and physiologically acceptable water-soluble substance as long
as they are solid substances (in the forms of prepared products) at body
temperatures of animals or humans to which they may be administered. One
water-soluble substance or a combination of 2 or more water-soluble
substances can also be used. Specifically, one or a plurality of
water-soluble substances can be selected from the group consisting of
synthetic polymers (e.g., polyethylene glycol and
polyethylenepolypropyleneglycol), sugars (e.g., sucrose, mannitol,
glucose, and sodium chondroitin sulfate), polysaccharides (e.g.,
dextran), amino acids (e.g., glycine and alanine), inorganic salts (e.g.,
sodium chloride), organic salts (e.g., sodium citrate) and proteins
(e.g., gelatin and collagen and a mixture thereof). Moreover, when a
water-soluble substance is an amphiphilic substance that can be dissolved
in both organic solvent and water, the solubility of the substance is
altered so as to have an effect of controlling the release of a
lipophilic remedy. Such an amphiphilic substance may contain one or a
plurality of substances selected from the group consisting of
polyethylene glycol or a derivative thereof, polyoxyethylene
polyoxypropylene glycol or a derivative thereof, and sugar ester of fatty
acid and sodium alkyl sulfate, and further specifically, polyethylene
glycol, polyoxystearate 40, polyoxyethylene, polyoxypropylene glycol,
sucrose ester of fatty acid, sodium lauryl sulfate, sodium oleate, and
sodium desoxycholate (deoxy sodium cholate (DCA)). However, the examples
are not limited thereto. Such a water-soluble substance may contain a
water-soluble substance having a kind of activity in vivo, such as a
low-molecular-weight drug, a peptide, a polypeptide, a protein, a
glycoprotein, polysaccharides or water-soluble drug (that is, antigenic
substance that is used as a vaccine.

[0041]Cyclodextrin is a water-soluble polysaccharide capable of forming
hollows, in which a water-insoluble drug can be contained. A drug can be
encapsulated within cyclodextrin using procedures known by persons
skilled in the art. For example, see ed., by Atwood et al., "Inclusion
Compounds" vol. 2 and vol. 3, Academic Press, NY (1984); Bender et al.,
"Cyclodextrin Chemistry," Springer-Verlag, Berlin (1978); Szeitli et al.,
"Cyclodextrins and Their Inclusion Complexes," Akademiai Kiado, Budapest,
Hungary (1982) and International Publication No. 00/40962.

[0042]A nanoparticle or a fine particle is a concentration core, which is
enclosed by a polymer shell (nanocapsule) or a nanosphere, in which a
solid or a liquid is dispersed in the entire polymer substrate. A general
method for preparing nanoparticles and fine particles is described in
Soppimath et al., J. Control Release (2001) 70(12): 1-20 which is
incorporated herein by reference in their entirety. Another example of
the polymer delivery vehicle that can be used herein is a block copolymer
micelle containing a drug that contains a hydrophobic core enclosed by a
hydrophilic shell. The micelle is generally used as a vehicle for a
hydrophobic drug and can be prepared as described in Allen et al.,
"Colloids and Surface B), Biointerfaces (1999) Nov. 16 (1-4): 3-27. A
polymer-lipid hybrid system comprises a polymer nanoparticle that is
enclosed by a lipid monolayer. Polymer particles function as a cargo
space for incorporation of a hydrophobic drug and a lipid monolayer
functions as a stable boundary between the hydrophobic core and the
external aqueous environment. As polymers, polycaprolactone,
poly(d,l-lactide), and the like can be used. A lipid monolayer is
generally composed of a lipid mixture. A suitable method therefor is
similar to that described in the above reference concerning polymer
nanoparticles. Derivatized single-chain polymer is a polymer that is
modified to be suitable for the formation of a polymer-drug conjugate via
covalent binding with a physiologically active substance. Various
polymers including polyamino acids, polysaccharides (e.g., dextrin or
dextran), and synthetic polymers (e.g.,
N-(2-hydroxypropyl)methacrylamide(HPMA) copolymer) are proposed as
polymers for the synthesis of polymer-drug conjugates. A preparation
method suitable therefor is described in detail in Veronese and Morpurgo,
I L Farmaco (1999) 54(8): 497-516, which is incorporated herein by
reference in their entirety.

[0043]In the description, "cell surface molecule" refers to an arbitrary
molecule that is present on the surface of a cell. Examples of such cell
surface molecule include, but are not limited to, lectins, cell adhesion
molecules, receptors, proteins, lipids, phospholipids, transmembrane
domains, and extracellular matrices.

[0044]In the description, "lectin" refers to a substance capable of
binding to a sugar chain of a cell membrane glycoconjugate (glycoprotein
and glycolipid) and having effects such as cell aggregation, division
induction, activation of functions, and cell damage. If a sugar chain is
assumed to be an information molecule transmitted from cells, it can be
said that a lectin is a receiving molecule. Cells or tissues having a
certain degree of properties have a pattern of lectin corresponding
thereto. Lectins realize infection, biophylaxis, immune, fertilization,
targeting target cells, cell differentiation, intercellular adhesion,
quality control of nascent glycoprotein, intracellular selective
transport, and the like. Lectins have a variety of sugar chain-binding
property and unique physicochemical characteristics such as rapid binding
and dissociation, so that the lectins are strictly controlled. Lectins
are also referred to as sugar chain-recognizing proteins. Plant lectins
have long been studied so that approximately 300 types of plant lectin
have already been known. Recently, animal lectins have also been actively
studied, so that novel lectins have been discovered one after another. A
wide variety of sugar chain-recognizing functions based on a lectin group
(approximately 100 types or more) of the major lectin family that is
present on animal cell membranes have been studied. In particular, the
functions as a receptor (an information-receiving protein or a target
molecule) that receives the structural information of a sugar chain
ligand varying in structure are attracting attention. In the description,
explanation is given based mainly on lectins, however, it is understood
that similar explanation is possible for cell surface molecules other
than lectins.

[0045]In the description, "ligand" refers to a substance that specifically
binds to a protein in biochemical fields. For example, substrates that
bind to enzymes, peptides, hormones, neurotransmitters, and the like that
bind to various receptor proteins (referred to as receptors) existing on
the cell membranes are referred to as ligands to their corresponding
proteins. Hence, in the case of this study, to use each type of lectin
protein (that functions as a type of receptor that is present on the
specific cell membranes of a target tissue) as a target molecule, a sugar
chain that is a ligand of the protein is introduced onto a liposome
surface, so that a DDS nanoparticle having an active targeting function
imparted thereto is prepared.

[0046]A lectin is present on the cell membrane surface and a sugar chain
that recognizes the lectin is present at the same time. In this case
cell-to-cell interaction takes place. Lectin-positive cells may interact
with sugar chain ligand-positive cells. A soluble glycoprotein may act on
lectin-positive cells and a soluble lectin may act on sugar chain
ligand-positive cells.

[0047]The binding dissociation constant (KD) between a sugar chain
and a lectin ranges from approximately 10-3M to 10-6M. Binding
between a sugar chain and a lectin is far weaker than that of an antigen
antibody reaction (KD: 10-7 to 10-12). However, it is
important for intercellular adhesion and selection to determine whether
or not binding should be performed based on weak binding. Therefore, in a
preferred embodiment, a cell surface molecule having binding dissociation
constant at such a level can have an advantage, but the example is not
limited thereto.

[0048]Sugar chains and lectins may have a plurality of binding sites in
one molecule or may be present as molecular assemblies. Although their
weak binding force, they are complemented by polyvalency (10-8M to
10-10M). Because of their strict binding specificity, they can be
used for strict delivery of a substance to a specific tissue and the
like.

[0049]When the present invention is implemented, in vitro affinity of
delivery vehicle candidates for a cell surface molecule such as a lectin
associated with a desired site is measured upon screening. It has been
discovered that a delivery vehicle having a specific tendency in terms of
in vitro affinity is a delivery vehicle preferred in vivo. This is
referred to as "rolling model" in the description.

[0050]"Rolling model" of the present invention is based on a finding that
a delivery vehicle having a low inhibitory concentration of strong
binding (in which n of ICn is relatively small) and having a high
inhibitory concentration of weak binding (in which n of ICn is relatively
large) exerts good delivery to target organs or tissues. The rolling
model has been completed by a discovery that a substance showing a gentle
concentration curve in the vicinity of a threshold near the boundary
between strong binding and weak binding is preferable as a delivery
vehicle. A theory has thus been discovered that such a substance does not
firmly bind in a target organ or tissue but should have a property of
rolling while appropriately binding. When such substances have been
actually screened for and tests have been conducted in vivo, all the
substances have achieved in vivo preferable delivery results. Therefore,
the present invention provides revolutionary assay system and screening
system by which whether a delivery substance can show a preferable
delivery result in a target organ or tissue can be determined by
performing simple assay in vitro.

[0051]Therefore, in the description, "strong binding" refers to binding in
which n of ICn is relatively small and specifically refers to binding
with approximate ICn (here, n is typically smaller than approximately
30). Strong binding may be appropriate for testing relatively strong
binding force (for example, antigen antibody reaction may be included).
Here, "strong binding inhibitory concentration (IC)" refers to an
inhibitory concentration with low percentage of inhibition.

[0052]Therefore, in the description, "weak binding" refers to binding in
which n of ICn is relatively large and specifically refers to binding
with approximate ICn (here, n is typically larger than approximately 31).
Weak binding may be appropriate for testing relatively weak binding force
(for example, binding that is almost non-specific binding is included
herein). Here, "weak binding inhibitory concentration (IC)" refers to an
inhibitory concentration with high percentage of inhibition.

[0053]These strong binding and weak binding are also assumed to vary
depending on cell surface molecules such as lectins. It is understood
that persons skilled in the art can appropriately determine n of ICn
depending on a system to be used.

[0054]Preferably through combination of index numbers of the strong
binding and weak binding, the present inventors have succeeded in
discovery of an appropriate delivery vehicle for rolling with the use of
an in vitro system.

[0055]According to the present invention, the thus selected delivery
vehicle does not firmly bind to tissues and makes it possible to
efficiently release active ingredients such as a drug that is delivered
together with the vehicle into a target. Hence, the delivery vehicle can
achieve far more efficient delivery compared with an antigen antibody
reaction that is accompanied by firm binding to a tissue. With the
"rolling model" theory of the present invention, such a system that can
ideally achieve such delivery can be conveniently discovered.

[0056]No theoretical constraints are desired herein, however, three
technological elements of DDS can mainly exist: drug-releasing
technology, drug-targeting technology, and drug-absorption-controlling
technology. The drug-releasing technology includes "release control
technology" of dispersing a drug in a polymer matrix or the like so as to
release a given amount of drug over a long time and "effective release
technology" of encapsulating a protein formulation or the like that is
almost insoluble in water within a vehicle such as a liposome so as to
cause the effective expression in vivo of the medicinal effect.

[0057]Drug-targeting technology includes "active targeting technology" of
actively delivering a drug to affected parts with the use of a ligand or
an antibody that recognizes cancerous tissues and "passive targeting
technology" of binding a polyethylene glycol or the like to a drug so
that the drug that is injected into a blood vessel is not easily
metabolized in the liver and the like and the drug is circulated in vivo
for a long time, thereby causing accumulation of the drug in affected
parts such as cancer.

[0058]The drug-absorption-controlling technology includes "drug
introduction technology" of causing absorption of a drug via mucous
membrane or skin and "gene introduction technology" of introducing a gene
into cells, so as to treat the disease, which is employed for gene
therapy and the like.

[0059]Among them, it has been revealed that an optimum combination of the
active-targeting technology and the effective release technology can be
conveniently discovered with the use of the rolling model of the present
invention. It can be said that this is a special effect that has been
unable to be discovered by conventional methods.

[0060]A typical example of a cell surface molecule that can be used in the
present invention is a lectin. Examples of such a lectin include, but are
not limited to, selectin (e.g., L-selectin, E-selectin, and P-selectin),
lectins involved in intracellular transport and selection of
glycoproteins (e.g., a mannose-6-phosphate receptor, calnexin,
calreticulin, ERGIC-53, and VIP53), cytokines (e.g., interleukins,
interferons, and growth factors), and galectin.

[0061]Among lectins, "selectin" in the description refers to a
transmembrane type glycoprotein that is of a group of cell adhesion
molecules that recognize sugar chains and has the N-terminus
extracellularly and the C-terminus intracellularly. These molecules of
this type have extracellularly, from the terminus in order, lectin domain
(L) that recognizes sugar chains in a Ca2+-dependent manner, EGF
(epidermal growth factor)-like domain (E) that has three disulfide bonds,
and complement-binding domain (C) that has homology with a
complement-binding protein. These molecules may also be referred to as
LECAM (lectin-type cell adhesion molecules) or as an LECAM family. There
are at least three types of molecules: L-selectin (LECAM-1) that is
expressed by leukocytes, E-selectin (ELAM-1 and LECAM-2) that is
expressed by activated vascular endothelial cells, P-selectin (GMP-140
and LECAM-3) that is expressed by activated blood platelet and activated
vascular endothelial cells.

[0062]L-selectin is expressed constitutively in most leukocytes. As
ligands for L-selectin, GlyCAM-1 (glycosylatkon-dependent cell adhesion
molecule), CD34, MAdCAM-1 (mucosal addressin cell adhesion molecule), and
the like are known. L-selectin achieves intercellular adhesion via its
binding to sialyl-6-sulfo Lex and is involved in homing phenomenon by
which lymphocytes in bloodstream assemble in specific lymphoid tissues.

[0063]E-selectin is often expressed on inflamed vascular endothelial
cells, by which granulocytes, monocytes, and the like assemble at the
inflammation sites. When vascular endothelial cells are stimulated with
interleukin1 (IL-1), tumor necrosis factor α (TNF-α),
endotoxin, and the like, expression of E-selectin is induced within
several hours. Therefore, it is understood according to the rolling
theory of the present invention, in vitro affinity for E-selectin is an
indicator for delivery to vascular endothelial cells, inflammation sites,
tumors, and the like.

[0064]P-selectin is contained in α granules of blood platelets and
Weibel-Palade bodies of endothelial cells. Degranulation takes place due
to stimulation with thrombin and then P-selectin is expressed after its
transfer to the cell surface. P-selectin is known to recognize the sialyl
LeX sugar chain of P-selectin glycoprotein ligand (PSGL-1) molecule
and N-terminal sulfated tyrosine residue together.

[0065]A mannose-6-phosphate receptor recognizes a structure in which a
phosphate group is added at position 6 of non-reduction terminal mannose
residue of a high-mannose type sugar chain. Such mannose-6-phosphate
receptor is present in the trans-Golgi network. This is a reason why
enzyme groups localized in lysosomes have specific sugar chains as labels
for selective transport.

[0066]The mannose-6-phosphate receptor includes two types:
Ca2+-independent receptor showing anti-affinity and
Ca2+-dependent receptor showing low affinity. The former receptor is
a 275-kDa transmembrane glycoprotein and the latter receptor is a 46-kDa
transmembrane glycoprotein.

[0067]Calnexin and calreticulin are types of molecular chaperone and are
lectins specifically binding to Glc1Man5-9GlcNAc sugar chain.

[0068]ERGIC-53 (ER-Golgi intermediate compartment) and VIP36 (vesicular
integral protein 36) are intracellular lectins containing sugar-binding
sites and calcium-binding sites. ERGIC-53 and VIP36 are present in a
region ranging from the endoplasmic reticulum to cis-Golgi and a region
ranging from the endoplasmic reticulum to the cell membrane,
respectively.

[0069]The relationships between lectins and organs can be explained as
follows. Various types of lectins (sugar chain-recognizing proteins) have
been studied as receptors existing on cell membrane surfaces of various
types of tissue in vivo, such as C-type lectins (e.g., selectin, DC-SIGN,
DC-SGNR, collectin, asialoglycoprotein receptor, and mannose-binding
protein), 1-type lectins (e.g., siglec), P-type lectins (e.g.,
mannose-6-phosphate receptor), R-type lectins, L-type lectins, M-type
lectins, and galectin. Sugar chains having various types of molecular
structure capable of binding to these lectins are attracting attention as
new DDS ligands.

[0070]The relationships between lectins and organs are as listed below, in
which the expression of lectins in human tissues has been revealed.

[0072]Regarding relationships between lectins and disease tissues,
expression of E-selectin, P-selectin, and the like in all the general
inflammatory diseases (e.g., encephalitis, chorioretinitis, pneumonia,
hepatitis, and arthritis) and diseases that continuously cause
inflammation (e.g., malignant tumor, rheumatism, cerebral infarction,
diabetes, and Alzheimer disease) is being elucidated. Moreover,
expression of various types of lectin including E-selectin, selectin,
galectin, siglec, and the like in cancer, brain diseases, cardiac
diseases, arteriosclerosis, and the like has been reported. A lot about
relationships between lectins and organs or diseases remains unknown and
is expected to be elucidated in the future.

[0073]When animal lectins are classified in terms of primary structure,
they can be classified into the following 14 types of family, for
example:

[0074]As the relationships between a great variety of animal lectins and
organs or diseases have been elucidated as described above, the
usefulness of the delivery vehicle (e.g., sugar-chain-modified liposome)
of the present invention in treatment and diagnosis of diseases will be
increased. Furthermore, the delivery vehicle can be applied to a broader
range of fields for treatment or diagnosis of diseases. Furthermore, the
delivery vehicle of the present invention is also useful as a reagent for
research for the purpose of elucidation of the biological significance of
a variety of animal lectins.

[0075]Research and development in the field of drug targeting system (DDS)
is classified into passive-targeting DDS and active-targeting DDS in
terms of targeting DDS. The passive-targeting DDS is a method for
controlling in vivo behavior via alteration of physicochemical properties
of a carrier (drug carrier), such as particle diameter or hydrophilicity.
The active-targeting DDS is a method for actively enhancing the tropism
for a target tissue through the use of a specific mechanism such as a
molecular recognition function added to the mechanism of the former
method. Research has been conducted concerning active-targeting to date.
As a result, many studies have been conducted concerning DDS
nanoparticles. For example, liposomes were prepared by binding various
types of ligand (e.g., antibody, transferrin, folic acid, and
monosaccharide) having molecular recognition functions to recognize
various types of cell surface molecule. However, most of these
nanoparticles bound to target cells in vitro (ex vivo), but were not
targeted in vivo to target tissues or cells expected to be targeted
(document 1 and document 2). For example, in vivo distribution of
liposomes having an anti-HER2 antibody bound as a ligand (the "anti-HER2"
antibody is an antibody against a protein referred to as "HER2" that is a
cell surface molecule on breast cancer cell surfaces) thereto and that of
liposomes having no such protein bound thereto were examined after their
injection via mouse tail vein (document 3). As a result, accumulation of
the former liposomes in cancer tissues was not improved because of the
binding of anti-HER2. Moreover, in vivo distribution of liposomes having
folic acid bound thereto and that of liposomes having no such acid bound
thereto were examined using a folic acid receptor (a cell surface
molecule of another cancer cell) as a target molecule, after their
injection via mouse tail vein (document 4). As a result, the accumulation
of the former liposomes in cancer tissues was not improved in spite of
binding of folic acid thereto. The target type of these two types of
ligand-bound liposome was not the type of cell surface molecule (1) in
FIG. 3, but was the type of cell surface molecule (2).

[0077]Examples of ligands that can be used in the present invention
include, but are not limited to, various types of sugar chain ligand
having molecular recognition functions to recognize various types of
lectin molecule and various types of ligand having molecular recognition
functions to recognize various types of cell surface molecule including
many lectin molecules. A target molecule of the DDS delivery vehicle of
the present invention is the type of lectin molecule (1) in FIG. 12, for
example. Such a target molecule can be used in combination with the type
of lectin molecule (2) if necessary. Furthermore, the type of lectin
molecule (1) in FIG. 13 can also be used as such a target molecule and
the lectin molecule (1) can also be used in combination with the type of
cell surface molecule (2) if necessary. Moreover, the type of cell
surface molecule (1) in FIG. 14 can also be used as such a target
molecule and the molecule (1) can also be used in combination with the
type of cell surface molecule (2) if necessary. Furthermore, the type of
cell surface molecule (1) in FIG. 15 can also be used as such a target
molecule and the molecule (1) can also be used in combination with the
type of lectin molecule (2) if necessary.

[0078]Currently, lectin molecules have been elucidated as listed in Table
1A below. Lectin molecules will become increasingly elucidated. It is
understood that persons skilled in the art having such knowledge will be
able to implement various embodiments based on descriptions given for the
present invention.

[0079]Cell surface molecules have been elucidated to date as listed in
Table 1B below. Cell surface molecules will become increasingly
elucidated. It is understood that persons skilled in the art having such
knowledge will be able to implement various embodiments based on
descriptions given for the present invention.

[Table 1B] Cell surface molecules that have been elucidated to
date(Document 3) http://www.hlda8.org/HLDAtoHCDM.htm

[0080]"Cytokine" that is used in the description is defined to be
interpreted in the broadest sense in the art. "Cytokine" refers to a
physiologically active substance that is produced from cells and acts on
the same or different cells. Cytokines are generally proteins or
polypeptides and have an effect of controlling immune response, an effect
of regulating the endocrine system, an effect of regulating the nervous
system, an anti-tumor effect, anti-virus effect, an effect of regulating
cell proliferation, an effect of regulating cell differentiation, and the
like. In the description, a cytokine may be in the form of protein or
nucleic acid or in another form. At the time when a cytokine actually
exerts an effect, the cytokine meant herein is generally in the form of
protein. Some of cytokines can be defined as lectins.

[0081]"Growth factor" or "cell growth factor" to be used in the
description refers to a substance that is used interchangeably in the
description and promotes or controls cell proliferation. Growth factors
are also referred to as Seicho inshi or Hatsuiku inshi. The effect of a
growth factor can be an alternative for the effect of a serum
macromolecular substance when the growth factor is added to vehicle upon
cell culture or tissue culture. It has been revealed that many growth
factors can function as factors for controlling cell differentiation in
addition to cell proliferation. Such growth factor can also be defined as
one of lectins.

[0083]Physiologically active substances such as cytokines and growth
factors generally undergo the phenomenon of functional redundancy
(redundancy). Hence, even cytokines or growth factors that are known as
other names and are known to have other functions, can be used in the
present invention, as long as they have the activities of physiologically
active substances that are used in the present invention. Moreover,
cytokines or growth factors can be used in preferred embodiments of the
treatment methods or remedies of the present invention, as long as they
have activities preferred in the description.

[0084]"Galectin" is a generic term for lectins binding to β
galactoside. Galectins are known to form a group of proteins having
homology with the amino acid sequence of sugar chain-recognizing domains
(CRDs). At least 14 types of galectin have been identified as having a
molecular weight ranging from 14 kDa to 36 kDa and having 1 to 2 types of
CRD. Galectins are soluble proteins having no membrane binding regions,
but binding to various ligands in vivo. Galectins are involved in
substitution of the hydroxyl group of β-galactose and in binding
specificity to aglycone molecules. The presence of galectins has been
confirmed in cytoplasms, nuclei, cell membranes, extracellular matrices,
and the like. It is said that galectins are involved in cell-to-substrate
interaction, cell proliferation control, control of RNA transport from
the nucleus, cytoskeleton formation, apoptosis induction or suppression,
neural induction, and the like. Galectins are classified into four types
in terms of structure: galectins 1, 2, and 7 that are dimers of the same
type; galectin 5 that is a monomer; galectins 4, 6, 8, and 9 that are
single-chain polypeptides each having two binding regions linked via
linker peptides; galectin 3 that is a protein having a single binding
region and a short N-terminus. The expression and distribution of
galectins in tissues differ depending on galectin types. Galectins are
distributed tissue-specifically. In humans, galectin 1 is expressed in
the skeletal muscle, neurons, kidney, placenta, and thymus gland,
galectin 2 is expressed in tumors in the liver, galectin 3 is expressed
by activated macrophages, eosinophils, neutrophils, mast cells, the small
intestine, the epithelium of a respiratory organ, and sensory neurons,
galectin 4 is expressed in the intestine or the epithelium of the oral
cavity, galectin 5 is expressed by erythrocytes or reticulum cells,
galectin 6 is expressed on the epithelium of the intestinal tract,
galectin 7 is expressed by keratinocytes, galectin 8 is expressed in the
lungs, liver, kidney, heart, and brain, and galectin 9 is expressed in
the liver, small intestine, kidney, lymphoid tissue, lungs, cardiac
muscle, and skeletal muscle.

[0085]Lectins that are specifically distributed will be described below.

[0086]A mannose-6-phosphate receptor is distributed in the trans-Golgi
network of each cell and is known to recognize a high-mannose type sugar
chain on a lysosome enzyme as a ligand.

[0087]Calnexin is distributed in the endoplasmic reticulum and is known to
recognize a nascent glycoprotein that is an α glucosylation type N
saccharine as a ligand.

[0088]Calreticulin is distributed in the endoplasmic reticulum and is
known to recognize a nascent glycoprotein that is an α
glucosylation type N saccharine as a ligand.

[0089]ERGIC-53 is distributed in a region ranging from the endoplasmic
reticulum to cis-Golgi and is known to recognize a mannose-containing
sugar chain as a ligand.

[0090]VIP36 is distributed in a region ranging from the endoplasmic
reticulum to the cell membrane and is known to recognize a high-mannose
type sugar chain as a ligand.

[0091]Galectins are distributed tissue-specifically as described above and
are known to recognize a β-galactose-type sugar chain as a ligand.

[0092]Siglec1 (sialoadhesin) is distributed in macrophages and is known to
recognize Siaα2-3Gal as a ligand.

[0093]Siglec2 (CD22) is distributed in lymphocytes (B cells) and is known
to recognize Siaα-2-6Galβ1-4GlcNAc as a ligand.

[0094]Siglec3 (CD33) is distributed in myeloid cells and is known to
recognize Siaα-2-3Gal as a ligand.

[0095]Siglec4a (MAG) is present in peripheral nerves and is known to
recognize Siaα2-3Gal as a ligand.

[0096]Siglec5 (myelin protein) is present in monocytes and is known to
recognize a sialic acid-containing sugar chain as a ligand.

[0097]N-CAM is distributed in peripheral nerves and is known to recognize
a high-mannose type sugar chain as a ligand.

[0098]Po (mammalian peripheral myelin, an intercellular adhesion factor
existing on mature Schwann cells) is distributed in peripheral nerves and
is known to recognize an HNK-1 antigen as a ligand.

[0099]L-selectin is distributed in leukocytes and is known to recognize a
sialyl 6-sulfo LeX on vascular endothelial cells as a ligand.

[0100]E-selectin is present in vascular endothelial cells and is known to
recognize a sialyl LeX of lymphocytes as a ligand.

[0101]P-selectin is present in vascular endothelial cells and is known to
recognize sialyl LeX and tyrosine sulfate on lymphocyte PSGL-1 as
ligands.

[0102]A mannose binding protein is present in lymphocytes (natural killer
cells) and is known to recognize N-sugar chain as a ligand.

[0103]An asialo-glycoprotein receptor is distributed in the liver and
recognizes triantenna and tetraantenna complex-type sugar chains of
proteins such as serum as ligands.

[0104]A macrophage mannose receptor is distributed in macrophages and is
known to recognize a mannose-containing sugar chain as a ligand.

[0105]Antithrombin (blood coagulation factor) is present in blood and is
known to recognize heparin as a ligand.

[0106]FGF is distributed in blood and is known to recognize heparan
sulfate as a ligand.

[0107]Interleukin2 (IL-2) is distributed in blood and is known to
recognize a high-mannose type sugar chain on an IL-2 receptor α
subunit as a ligand.

[0108]Interleukin1α (IL-1α) is distributed in blood and is
known to recognize an asialo-biantenna sugar chain as a ligand.

[0109]Interleukin1β (IL-1β) is distributed in blood and is known
to recognize GPI anchor sugar chain glycolipid GM4 as a ligand.

[0110]Interleukin3 (IL-3) is distributed in blood and is known to
recognize heparan sulfate as a ligand.

[0111]Interleukin6 (IL-6) is distributed in blood and is known to
recognize an HNK-1 antigen as a ligand.

[0112]Interleukin7 (IL-7) is distributed in blood and is known to
recognize a sialyl Tn antigen as a ligand.

[0113]Tumor necrosis factor α (TNF-α) is distributed in blood
and is known to recognize a mannose-containing sugar chain as a ligand.

[0114]"In vitro affinity" for a lectin in vitro in the description can be
determined by measuring affinity (e.g., Example 7 in the description) for
a lectin associated with a target site (e.g., E-selectin in the case of
an inflammation site) in an in vitro experiment. The affinity can be
measured by an inhibition experiment using lectin-immobilized microplates
as described in Yamazaki, N. (1999) Drug Delivery System, 14, 498-505,
for example. Specifically, a lectin (e.g., E-selectin; R&D Systems Co.,
U.S.A.; Various lectins can be used herein depending on target organs.)
is immobilized on a 96-well microplate. Various types of sugar
chain-bound liposome complex (protein amounts: 0.01 μg, 0.04 μg,
0.11 μg, 0.33 μg, and 1 μg) varying in concentration were added
together with 0.1 μg of biotinylated and fucosylated fetuin as a
comparative ligand to the lectin-immobilized plate, followed by 2 hours
of incubation at 4° C. After 3 times of washing with PBS (pH 7.2),
horseradish peroxidase (HRPO)-conjugated Streptavidin is added and then
incubation is performed for 1 hour at 4° C. After 3 times of
washing with PBS (pH 7.2), a peroxidase substrate is added and then the
resultant is allowed to stand at room temperature. Absorbance at 405 nm
can be measured using a microplate reader (Molecular Devices Corp.,
U.S.A.).

[0115]Binding constant can be expressed as ICn ("n" is an arbitrary number
between 1 and 99, such as 10, 20, 30, 40, 50, and 60; unit: concentration
(M)). The calculation method for ICn to be used herein is as described
below. "IC" used herein indicates inhibitory concentration.

[0116]Binding index number (proportion) is measured at various
concentrations. For example, the values of sample LY-1 were measured.

[0117]Table 1 shows the results. Here, in vitro lectin binding activity
was determined by an inhibition experiment using lectin-immobilized
microplates according to a standard method (Yamazaki, N. (1999) Drug
Delivery System, 14, 498-505). Specifically, a lectin (e.g., E-selectin;
R&D Systems Co., U.S.A.; Various lectins can be used herein depending on
target organs.) was immobilized on a 96-well microplate. Various types of
sugar chain-bound liposome complex (protein amounts: 0.01 μg, 0.04
μg, 0.11 μg, 0.33 μg, and 1 μg) varying in concentration were
added together with 0.1 μg of biotinylated and fucosylated fetuin as a
comparative ligand to the lectin-immobilized plate, followed by 2 hours
of incubation at 4° C. After 3 times of washing with PBS (pH 7.2),
horseradish peroxidase (HRPO)-conjugated Streptavidin was added and then
incubation was performed for 1 hour at 4° C. After 3 times of
washing with PBS (pH 7.2), a peroxidase substrate was added and then the
resultant was allowed to stand at room temperature. Absorbance at 405 nm
was measured using a microplate reader (Molecular Devices Corp., U.S.A.).
Biotinylation of fucosylated fetuin was performed by performing treatment
with a sulfo-NHS-biotin reagent (Pierce Co., U.S.A.) and then
purification using Centricon-30 (Amicon Co., U.S.A.). HRPO-conjugated
Streptavidin was prepared by oxidizing HRPO and binding of Streptavidin
via reductive amination using NaBH3CN. The measurement results were
processed and calculated as described below.

TABLE-US-00003
TABLE 1
Values of sample LY-1 and IC50 at each concentration
Concentration
0.01 0.04 0.11 0.33 1
Average value of sample LY-1 0.144 0.142 0.126 0.110 0.073
Ratio of average value of sample 0.739 0.715 0.562 0.414 0.060
LY-1*
Ratio of y coordinate of IC50 series 0.500 0.500 0.500 0.500 0.500
*"Ratio of the average value of sample LY-1" or "Ratio of the y coordinate
of IC50 series is the ratio of the same with respect to 1 (a difference
between a "hot" value and a "cold" value of Control is determined to be
"1.")

TABLE-US-00004
TABLE 2
Control
Hot Cold
0.171 0.067

[0118]Therefore, when the ratio of the average value of sample LY-1 is
"W," the average value of sample LY-1 is "X," the value of "hot" is "Y,"
the value of "cold" is "Z," and the calculation formula can be expressed
as

W=(X-Z)/(Y-Z)×100.

[0119]FIG. 2 shows Graph 1 in which the values of sample LY-1 obtained
based on Table 1 and Table 2 and IC50 series are plotted.

[0120]Graph 1 was prepared based on Table 1. X axis is expressed using a
logarithm scale. Each point on the line graph represents the ratio of the
average measured value at each concentration (horizontal axis) of sample
LY-1. Values of control differ depending on samples. To facilitate
comparison, the longitudinal axis of the graph represents the ratios when
a difference between the value of "hot" and the value of "cold" is
determined to be "1." X coordinate at the intersection point between the
graph of Sample LY-1 and the graph of IC50 series represents the value of
IC50. The intersection point exists on the straight line including
coordinate 1 (0.11, 0.562) and coordinate 2 (0.33, 0.414) and is
represented by formula y=-0.673x+0.636. When y=0.5 (formula for IC50
series), X coordinate at the intersection point of two straight lines is
0.202. This value is divided by the molecular weight of the protein,
69000, and then the result is further divided by 300 (the number of
protein per liposome). Thus 9.76E-09 is obtained.

[0121]These calculations can be automated using a computer program.

[0122]In the description, "in vivo affinity" refers to affinity for the
destination to which a delivery vehicle is actually delivered in vivo. In
vivo affinity can be determined by examining the biological dynamic state
of the delivery vehicle that is transferred to each organ. As a specific
example, in vivo affinity can be examined by administering a liposome via
oral or intravenous administration and then evaluating its accumulation
in each mouse organ. After intravenous injection or oral administration,
all organs are each excised. Each organ is prepared as a tissue
homogenate using 1% Triton X solution and HG30 homogenizer (Hitachi Koki
Co., Ltd.). Liposomes contained in tissue homogenates are extracted using
100% methanol and chloroform. The amount of a liposome is measured as
follows. The fluorescence intensity of FITC bound to the liposomes is
measured using a fluorescent microplate reader Biolumin 960 (Molecular
Dynamics), followed by measurement using excitation at 490 nm and
emission at 520 nm. The results obtained by this method can also be
represented by numerical figures, but can also be expressed comparatively
such that evaluation can also be made using +++, ++, +, -, and the like.

[0123]In the present invention, in vitro affinity determined by in vitro
assay using a cell surface molecule such as a specific lectin is
represented by n % inhibitory concentration (ICn; herein, "n" ranges from
0 to 100). The present invention is partially based on the finding that
the thus obtained numerical value correlates unexpectedly with in vivo
affinity (delivery specificity). As a result, based on the rolling model,
it has become possible to efficiently, conveniently, and precisely design
a delivery vehicle. Such a simple design of a delivery vehicle has been
impossible with the use of conventional technology and has been unknown.
The present inventors have established the above theory and completed the
present invention by examining and systematically studying several
hundred delivery vehicle candidates.

[0124]Furthermore, as a result of systematic studies, the present
inventors have discovered that ideal in vivo affinity can be predicted
with high probability by: measuring in vitro affinity at least one strong
binding IC in which n of ICn is smaller than a branch point that is a
numerical figure between IC35 and IC30 (e.g., between approximately IC31
and approximately IC30) and measuring in vitro affinity at least one weak
binding IC in which n of ICn is larger than the branch point; and
comparing the results in a comprehensive manner. As a result, it has been
demonstrated that a delivery vehicle (e.g., sugar-chain-modified
liposome) showing a low inhibitory concentration at the strong binding IC
and a high inhibitory concentration at the weak binding IC exerts high in
vivo affinity. No theoretical constraints are desired herein. It is
predicted that a delivery vehicle exerting such properties has preferred
characteristics in view of "rolling."

[0125]Realization of active targeting that involves actively delivering a
desired substance has been conventionally attempted using molecules
having high specificity to molecules existing in target cells. It has
been thought that the higher the binding nature, the more sufficient,
selective, and efficient delivery can be achieved. However, it has been
increasingly revealed that active targeting based on such idea is
unsuccessful. It has been revealed by the present invention that this may
be because, although no theoretical constraints are desired, excessively
high binding nature causes a delivery vehicle to remain bound on the
target so that the molecule to be delivered cannot be efficiently
delivered. When competitive inhibition takes place at a low concentration
at both strong binding IC and weak binding IC, the binding in this case
is thought to have the highest specificity. However, the resulting in
vivo affinity is not so high in most cases. Therefore, strong binding is
not always preferred. Rather, it is concluded that a delivery vehicle
showing a low concentration at the strong binding IC, but a high
concentration at the weak binding IC makes it possible to perform rolling
at its target site and efficiently deliver a substance to be delivered.

[0126]Preferably, the measurement of in vitro affinity of the present
invention comprises measurement at a strong binding IC that is at least
one between IC30 and IC10 and measurement at a weak binding IC that is at
least one between IC40 and IC60. In measurement at a strong binding IC
that is at least one between IC30 and IC10, ICn within the range may be
arbitrarily selected. Similarly, in measurement at a weak binding IC that
is at least one between IC40 and IC60, ICn within the range may be
arbitrarily selected.

[0127]In an embodiment, the measurement of in vitro affinity comprises
measurement at a strong binding IC that is approximately IC30 or less.
The selection comprises selecting a candidate showing a low inhibitory
concentration at the strong binding IC. In this case, a delivery vehicle
that enables good rolling; that is, has good in vivo affinity can be
identified with at least a constant probability. Preferably, the
measurement of affinity comprises measurement at a strong binding IC that
is approximately IC31 or less. A candidate showing an inhibitory
concentration of 10-9M or less typically at the strong binding IC is
selected. Here, "binding of less than IC30" means that "n" of ICn has a
numerical figure that is smaller than 30. In contrast, "binding of IC31
or more" means that "n" of ICn has a numerical figure that is 31 or more.

[0128]In a preferred embodiment, the above selection is made when the
inhibitory concentration at IC30 is 10-9M or less, the inhibitory
concentration at IC20 is 10-9M or less, and the inhibitory
concentration at IC10 is 10-9M or less.

[0129]In another embodiment, the measurement of in vitro affinity of the
present invention comprises measurement at a weak binding IC that is
approximately IC31 or more. With this selection, a candidate showing a
high inhibitory concentration at the strong binding IC is selected.
Preferably, the measurement of in vitro affinity comprises measurement at
a weak binding IC that is approximately IC31 or more. A candidate showing
an inhibitory concentration of 10-9M or more at the weak binding IC
is selected.

[0130]In a preferred embodiment, the selection is made when the inhibitory
concentration at IC60 is 10-9M or more, the inhibitory concentration
at IC50 is 10-9M or more, and the inhibitory concentration at IC40
is 10-9M or more.

[0131]In a preferred embodiment, the measurement of in vitro affinity of
the present invention comprises measurement at a strong binding IC that
is approximately IC30 or less and measurement at a weak binding IC that
is approximately IC31 or more. The above selection is characterized by
selecting a candidate showing a low inhibitory concentration at the
strong binding IC and a high inhibitory concentration at the weak binding
IC.

[0132]In a further preferred embodiment, the measurement of in vitro
affinity of the present invention comprises measurement at a strong
binding IC that is approximately IC30 or less and measurement at a weak
binding IC that is approximately IC31 or more. The above selection is
characterized by selecting a candidate showing an inhibitory
concentration of 10-9M or less at the strong binding IC and
inhibitory concentration of 10-9M or more at the weak binding IC.

[0133]In a further preferred embodiment, the measurement of in vitro
affinity of the present invention comprises measurement at a strong
binding IC that is approximately IC30 or less. The above selection is
characterized by selecting a candidate showing a low inhibitory
concentration at the strong binding IC. Here, the selection is further
characterized by satisfying at least one condition selected from the
group consisting of: a condition in which the inhibitory concentration at
IC60 is 10-9M or more, a condition in which the inhibitory
concentration at IC50 is 10-9M or more, a condition in which the
inhibitory concentration at IC40 is 10-9M or more, a condition in
which the inhibitory concentration at IC30 is 10-9M or less, a
condition in which the inhibitory concentration at IC20 is 10-9M or
less, and a condition in which the inhibitory concentration at IC20 is
10-9M or less.

[0134]In a preferred embodiment, as shown in a graph showing
concentration-inhibition % curves in FIG. 11, a possible advantageous
form in the rolling model has a gentle curve as represented by the solid
line. It was demonstrated by the present invention that possession of a
curve represented by a dotted line (e.g., antigen antibody reaction)
results in failed rolling and unsuccessful selective delivery.

[0135]A measurement method of in vitro affinity for determination of the
above numerical figures is performed by competitive inhibition assay,
noncompetitive inhibition assay, binding assay, or the like.

[0136]According to the present invention, once a preferred delivery
vehicle is determined in vitro, the delivery vehicle can then be
generated based on the composition. At this time, a substance desired to
be delivered (e.g., a pharmaceutical composition) can be contained in the
delivery vehicle.

[0137]Here, when the composition of a preferred delivery vehicle is
unknown, the composition can be determined according to need. As a method
for determination of such composition, an arbitrary method known in the
art can be employed. When the composition is analyzed, a method for
preparing a delivery vehicle having the composition can be determined.
For determination of such a preparation method, WO2002/081723, JP Patent
Publication (Kokai) No. 9-31095 A (1997), JP Patent Publication (Kokai)
No. 11-42096 A (1999), JP Patent Publication (Kokai) No. 2004-180676 A,
and Kenichi Hatanaka, Shinichiro Nishimura, Tatsuro Ohuchi, and Kazukiyo
Kobayashi (1997) Science and Engineering of Sugar (To-shisu no kagaku to
kogyo), Kodansha Ltd., Tokyo, Japan, and the like can be referred.
According to the present invention, it has been revealed that when a
delivery vehicle is a sugar-chain-modified liposome, not only the
composition, but also the sugar chain type and density play important
roles. Therefore, in a preferred embodiment, analysis of the composition
can comprise analysis of the sugar chain types and densities of the
sugar-chain-modified liposome. Once the type and density of a sugar chain
are determined, persons skilled in the art can determine a method for
producing a sugar-chain-modified liposome according to the techniques
described for the present invention. An example of such a production
method involves performing, upon generation of a sugar-chain-modified
liposome, a reaction of sugar chains (the types and the amounts of which
are determined based on the thus determined composition) under conditions
adequate for binding to a liposome. Preferably, a linker can be used
herein. As a linker, a protein such as albumin can be used, for example.
A liposome can be hydrophilized, according to need.

[0138]In a preferred embodiment, the method of the present invention
further comprises the step of confirming the in vivo dynamic state of the
thus selected delivery vehicle.

[0139]In another aspect, a method for producing a delivery vehicle by
which delivery to undesired sites is not performed is provided according
to the present invention, which is analogous to the method for delivery
to desired sites. Such production method comprises the steps of:

A) measuring in vitro affinity of candidate delivery vehicles for a cell
surface molecule such as a lectin associated with an undesired site;
andB) selecting a delivery vehicle having in vitro affinity corresponding
to non-delivery to the undesired site.

[0140]Alternatively, the production method can be a method that comprises
the steps of:

A) measuring in vitro affinity of candidate delivery vehicles for a cell
surface molecule such as a lectin associated with an undesired site;B)
selecting a delivery vehicle having in vitro affinity corresponding to
non-delivery to the undesired site and analyzing the composition of the
selected delivery vehicle; andC) generating the selected delivery vehicle
based on the composition.

[0141]In another aspect, the present invention provides a method for
producing a delivery vehicle for achieving specific delivery. The
production method comprises the steps of:

A) measuring in vitro affinity of candidate delivery vehicles for a cell
surface molecule such as a lectin associated with a site to which
specific delivery is performed;B) measuring in vitro affinity of the
candidate delivery vehicles for a cell surface molecule such as a lectin
associated with a site to which specific delivery is not performed; andC)
selecting a delivery vehicle having in vitro affinity corresponding to
delivery to the desired site and corresponding to non-delivery to the
undesired site.

[0142]Alternatively, the method can be a method that comprises the steps
of:

A) measuring in vitro affinity of candidate delivery vehicles for a cell
surface molecule such as a lectin associated with a site to which
specific delivery is performed;B) measuring in vitro affinity of the
candidate delivery vehicles for a cell surface molecule such as a lectin
associated with a site to which specific delivery is not performed;C)
selecting a delivery vehicle having in vitro affinity corresponding to
delivery to the desired site and corresponding to non-delivery to the
undesired site and analyzing the composition of the selected delivery
vehicle; andD) generating the selected delivery vehicle based on the
composition.

[0143]The thus produced delivery vehicle is also encompassed within the
scope of the present invention.

(Delivery Method)

[0144]In another aspect, the present invention provides a method for
delivering a biological factor to a target site of a subject who needs
the biological factor. This method comprises the step of: administering
the sugar-chain-modified liposome of the present invention via oral
administration, in which the sugar-chain-modified liposome contains an
effective dose of the biological factor. As the sugar-chain-modified
liposome, such a liposome in an arbitrary form as describe above
(sugar-chain-modified liposome) can be used.

(Specific Explanation of Delivery Vehicle)

[0145]In the description, "sugar chain" refers to a compound composed of
one or more sugar units (monosaccharides and/or derivatives thereof)
linked together. When two or more sugar units are linked, sugar units are
each bound via dehydration and condensation due to glycosidic linkage.
Examples of such sugar chains include, but are not limited to, a wide
range of sugar chains, in addition to polysaccharides (glucose,
galactose, mannose, fucose, xylose, N-acetylglucosamine,
N-acetylgalactosamine, sialic acid, and their complexes and derivatives
thereof) contained in vivo, sugar chains degraded or induced from complex
biomolecules (e.g., degraded polysaccharide, glycoprotein, proteoglycan,
glycosaminoglycan, and glycolipid). Therefore, in the description, "sugar
chain" can be used interchangeably with "polysaccharide," "sugar," or
"carbohydrate." Furthermore, unless otherwise particularly noted, "sugar
chain" in the description may refer to both a sugar chain and a
sugar-chain-containing substance. A typical sugar chain is a substance
composed of approximately 20 types of monosaccharide linked to form a
chain (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine,
N-acetylgalactosamine, and sialic acid, and complexes and derivatives
thereof) that is bound to intracellular and extracellular proteins or
lipids in vivo. The functions of such a sugar chain differ depending on
the monosaccharide sequences. Furthermore, sugar chains are generally
branched in a complicated manner. It is estimated that several hundred or
more types of sugar chain varying in their structures are present in a
human body. Furthermore, it is thought that there are several tens of
thousands or more of types of useful sugar chain structure in a human
body. It is thought that such sugar chain structures are involved in
higher functions exerted by proteins or lipids in vivo, such as a
function of recognizing molecules and a function of recognizing cells,
which are exerted between cells. Most of such mechanism remains
unelucidated. Sugar chains are attracting attention in the field of
current life science as the 3rd life chain following nucleic acids
and proteins. In particular, the functions of sugar chains as ligands
(information molecules) for cell recognition are expected and the
application of such sugar chains to development of highly functional
materials is studied.

[0146]In the description, "sugar" or "monosaccharide" refers to
polyhydroxy aldehyde or polyhydroxy ketone containing at least one
hydroxy group and at least one aldehyde group or ketone group and
composes a basic unit of a sugar chain. In the description, sugar is also
referred to as carbohydrate and both terms can be used interchangeably.
In the description, when particularly mentioned, "sugar chain" refers to
a chain or a sequence composed of one or more sugars linked. "Sugar" or
"monosaccharide" refers to one unit composing a sugar chain.

[0147]"Sugar" or "monosaccharide" in which n=2, 3, 4, 5, 6, 7, 8, 9, and
10 are each referred to as diose, triose, tetrose, pentose, hexose,
heptose, octose, nonose, and decose. In general, they correspond to
aldehyde or ketone of chain polyhydric alcohol. The former is referred to
as aldose and the latter is referred to as ketose. In the present
invention, "sugar" or "monosaccharide" in any forms can be used.

[0148]Naming of sugars to be described in the present invention is
performed according to the general nomenclature. Examples are as follows:

galactose is named

##STR00001##

Gal;

[0149]N-acetylgalactosamine is named

##STR00002##

GalNAc;

[0150]mannose is named

##STR00003##

Man;

[0151]glucose is named

##STR00004##

Glc;

[0152]N-acetylglucosamine is named

##STR00005##

GlcNAc;

[0153]fucose is named

##STR00006##

Fuc;

[0154]N-acetylneuraminic acid is named

##STR00007##

Neu5Ac;

[0155]ceramide is named

##STR00008##

Cer; L-serine

[0156]CH2(OH)CH(COOH)NH2 is named Ser. In addition, Cer is
generally classified as a lipid. However, in the description, Cer is
treated as a sugar unless otherwise particularly noted, since it meets
the definition for a type of sugar composing a sugar chain. Furthermore,
Ser is generally classified as an amino acid. However, in the
description, Ser is treated as a sugar unless otherwise particularly
noted, since it meets the definition for a type of sugar composing a
sugar chain. Two circular anomers are represented by α and β
or may also be represented by "a" or "b" because of reasons concerning
representation. Therefore, in the description, "α" and "a" can be
or "β" and "b" can be used interchangeably in terms of denotation of
anomers.

[0165]It should be noted in the description that denotational symbols,
nominal designation, abbreviated expressions (e.g., Glc), and the like
for sugars differ between a case when they are used to indicate
monosaccharides are indicated and a case when they are used to indicate
those in sugar chains. In a sugar chain, dehydration and condensation
take place between a sugar unit and a sugar unit (to which the former
sugar unit binds), so that the resultant is present as a result of
removing hydrogen or hydroxy groups from the other sugar unit. Therefore,
it is understood as follows. When the condensation codes of these sugars
are used to represent monosaccharides, all the hydroxy groups are
present. However, when such codes are used to represent those in a sugar
chain, a condition is indicated wherein hydroxy groups of a sugar unit
and hydroxy groups of the other sugar unit (to which the former sugar
unit binds) are subjected to dehydration and condensation so that oxygen
alone remains.

[0166]When a sugar is covalently bound to albumin, the reducing terminus
of the sugar is aminated, so that the sugar can bind to another component
such as albumin via the amine group. In this case, it should be noted
that the term indicates the one in which the hydroxyl group of the
reducing terminus is substituted with an amine group.

[0167]A monosaccharide generally forms a disaccharide or a polysaccharide
via glycosidic linkage. The orientation of linkage with respect to the
plane of the ring is denoted with "α" or "β." Specific carbon
atoms that form linkage between two carbons are also described.

[0168]In the description, a sugar chain is represented by:

##STR00009##

Therefore, for example, β glycosidic linkage between C-1 of galactose
and C-4 of glucose is represented by Galβ1, 4Glc.

[0169]Sugar chain branches are represented using parentheses.
Specifically, a sugar chain branch is denoted by locating a parenthese at
a position immediately left of a sugar unit to be bound. For example,
this is represented by:

##STR00010##

Such parenthesed part is represented by:

##STR00011##

Therefore, for example, when β glycosidic linkage is formed between
C-1 of galactose and C-4 of glucose and the C-3 of glucose and C-1 of
fucose form α glycosidic linkage, this is represented by
Galβ1, 4(Fucα1, 3)Glc. Monosaccharides are represented
basically by numbering (potential) carbonyl atomic groups with numbers
that should be as small as possible. Under the general standard of
organic chemical nomenclature, a case in which an atomic group with
superiority over a (potential) carbonyl atomic group is introduced into a
molecule, generally the above numbering is used for representation.

##STR00012##

[0170]An example of a sugar chain that is used in the description is a
sugar chain having at least one or at least two sugar units selected from
the group consisting of Gal, GalNAc, Man, Glc, GlcNAc, Fuc, Neu5Ac, and
Ser. The reason why a combination of two or more sugar units can be used
is, which is not theoretically constrained, that each of the above sugar
units has specificity to a cell surface molecule such as a lectin that is
localized in a target delivery site in tissues or cells and may be able
to exert its specificity even when they are mixed.

(Liposome)

[0171]As a delivery vehicle that can be used in the rolling method of the
present invention, any vehicle can also be used. A particularly preferred
example is a delivery vehicle using a liposome.

[0172]In the description, "liposome" generally means a closed vesicle that
is composed of a lipid layer and an internal aqueous layer, which
assemble to form a membrane. In addition to a typically used
phospholipid, cholesterol, a glycolipid, and the like can also be
incorporated. A liposome is a closed vesicle containing water
therewithin, so that it can also retain a water-soluble drug and the like
within the vesicle. Therefore, with the use of such a liposome, a drug, a
gene, or the like that is unable to pass through the cell membrane can be
delivered into the cell. Furthermore, such liposome also has good
biocompatibility, so that it is expected as a nanoparticular carrier
material for DDS.

[0173]Liposomes can be produced by any techniques known in the art. An
example of these techniques is a method that involves performing cholic
acid dialysis. Production is performed via cholic acid dialysis that
involves a) preparation of a mixed micelle of lipids and a surfactant and
b) dialysis of the mixed micelle. Next, in a preferred embodiment of the
sugar chain liposome of the present invention, it is preferable to use a
protein as a linker. Coupling of a glycoprotein (in which a sugar chain
is bound to the protein) to a liposome can be performed via the following
two-stage reaction: a) periodate oxidation of the ganglioside portion on
the liposome membrane; and b) coupling of a glycoprotein to the oxidized
liposome via reductive amination reaction. FIG. 1 shows an example of the
reaction flow. A glycoprotein containing a desired sugar chain can be
bound to a liposome with the use of such a technique. Furthermore, a
great variety of glycoprotein-liposome conjugates having desired sugar
chains can be obtained. It is very important to examine particle diameter
distribution to examine the purity and stability of liposomes. As methods
to be employed for such examination, gel filtration chromatography (GPC),
scanning electron microscopy (SEM), dynamic light scattering (DLS), and
the like can be employed. A type of liposome in which the molar ratio of
dipalmitoylphosphatidylcholine (DPPC), cholesterol, dicetylphosphate
(DCP), and ganglioside is 35:45:5:15 can be produced. In addition, the
liposome is stable even when it is stored at 4° C. for several
months. The in vivo stability of a liposome can be examined using mice. A
liposome is intravenously injected into a mouse. 3 hours later, blood is
collected and then serum is prepared. Ultrafiltration is performed using
a membrane with a pore size of 0.03 μm and then the liposome is
purified and collected. Based on the result of SEM observance performed
therefor, it can be confirmed that the liposome undergoes no
morphological changes before and after 3 hours of treatment and
collection in vivo.

[0180]Of these, phosphatidic acids, long-chain alkyl phosphates,
glycolipids, and cholesterols are desirably added as constituent lipids
since they have effects of elevating the stability of the liposome.
Examples of such a lipid composing the liposome of the present invention
include: one or more types of lipid (mole percentage: 0% to 30%) selected
from the group consisting of phosphatidyl cholines (mole percentage: 0%
to 70%), phosphatidyl ethanol amines (mole percentage: 0% to 30%),
phosphatidic acids, and long-chain alkyl phosphate; one or more types of
lipid (mole percentage: 0% to 40%) selected from the group consisting of
glycolipids, phosphatidyl glycerols, and sphingomyelins; and lipids
containing cholesterols (mole percentage: 0% to 70%). Preferably, a
glycolipid such as ganglioside is contained herein since it facilitates
binding of a linker such as albumin.

[0181]In a preferred embodiment, ganglioside can be contained in the
liposome of the present invention, a linker such as a peptide can be
bound thereto, and then a sugar chain can be bound to the resultant.

[0182]With the use of a glycolipid for preparation of a liposome, the
sugar-chain-modified liposome of the present invention containing a sugar
chain contained in the glycolipid as a constituent can be prepared.

(Sugar-Chain-Modified Liposome)

[0183]In an aspect, the present invention provides a sugar-chain-modified
liposome. Liposomes capable of sufficiently targeting desired target
cells or tissues in vivo have not been provided to date. The present
invention has an effect of making it possible to perform targeting that
has been impossible with the use of conventional DDS materials through
provision of a sugar-chain-modified liposome having tropism for desired
target cells or tissues in vivo. Further systematically, a sugar chain
having at least one structure selected from the group consisting of Gal,
GalNAc, Man, Glc, GlcNAc, Fuc, and Neu5Ac is bound to such
sugar-chain-modified liposome.

[0184]In the description, "sugar-chain-modified liposome" refers to a
substance containing a sugar chain and a liposome and preferably refers
to a liposome that is modified by direct or indirect binding of a sugar
chain thereto. Such a form in which a sugar chain is bound to a liposome
is specifically represented by:

[0185]When ganglioside is contained in a liposome, the
sugar-chain-modified liposome of the present invention is represented by

S-M-GS-L

(GS: sugar chain portion of ganglioside).

[0186]When this is more specifically described, in the description,
"proximal end of sugar chain to the liposome" of a sugar-chain-modified
liposome refers to a terminal portion of a sugar chain located most
proximal to the liposome. When a sugar chain is branched, all the
relevant terminal portions are referred to as proximal ends.

[0187]"Most proximal end of a sugar chain to the liposome" refers to a
sugar (monosaccharide) located most proximal to the liposome. Therefore,
in the description, it is understood that when "a sugar chain comprising
a di- or more (multi-) saccharide is contained at the proximal end of the
sugar chain to the liposome," the proximal end of the sugar chain to the
liposome contains, in addition to a saccharide (monosaccharide) at the
most proximal end of the sugar chain to the liposome, another saccharide
(monosaccharide that may be the same or different from the saccharide at
the most proximal end) contained in the above sugar chain comprising a di
or more (multi-) saccharide.

[0188]In the description, "distal end of a sugar chain to the liposome" of
a sugar-chain-modified liposome refers to a terminal portion of the sugar
chain, which is located most distal to the liposome. When a sugar chain
is branched, all the relevant terminal portions are referred to as distal
ends.

[0189]"Most distal end of a sugar chain to the liposome" refers to a
saccharide (monosaccharide) that is located most distal to the liposome.
Therefore, in the description, when "distal end of a sugar chain to the
liposome contains a sugar chain comprising a di- or more (multi-)
saccharide," it is understood that the distal end of the sugar chain to
the liposome contains, in addition to a saccharide (monosaccharide)
located at the most distal end of the sugar chain to the liposome,
another saccharide (monosaccharide that may be the same or different from
the saccharide at the most distal end) contained in the above sugar chain
comprising a di- or more (multi-) saccharide.

[0190]Examples adequate for the rolling model in the description are
listed in the following Table using liposome numbers (Each liposome No.
corresponds to the structure in the right column).

[0191]When used in the description, "modification (binding) density"
refers to an amount of a sugar chain that is used for preparation of a
sugar-chain-modified liposome. The term is conveniently used to represent
the density of a sugar chain (mg sugar chain/mg lipid) that binds per mg
of lipids in a liposome in the description. Although no theoretical
constraints are desired herein, regarding the binding density of the
sugar-chain-modified liposome of the present invention, it is empirically
known that the amount of a sugar chain that is used for preparation is
almost proportional to the density of a sugar chain bound to the
liposome. Therefore, in the description, unless otherwise particularly
noted, binding density is determined depending on the amount used upon
preparation. In vitro, for example, such density can be indirectly
determined using E-selectin. In the present invention, when a delivery
vehicle is a sugar-chain-modified liposome, tropism for a target delivery
site can be controlled via selection of the type and binding density of a
sugar chain to be bound to the liposome. Delivery vehicles and related
organ tropism are listed as follows.

Definition of tropism evaluation (++, +) is as described below.
Furthermore, evaluation (-) represents a negative result and NA represent
"not measured."

[0192]In addition, in the case of oral administration, the average value
of each liposome that is absorbed via the intestinal tract at 10 minutes
after administration is divided by the average value of the standard
liposome and the result is shown in table (average value of each
liposome/average value of the negative liposome).

[0193]This is represented as follows when the same Tris is used as a
standard for all cases.

[0195]In the present invention, a sugar-chain-modified liposome that is a
typical delivery vehicle can be produced by the following method.
Specifically, the method comprises the steps of: (a) providing a
liposome; (b) hydrophilizing the liposome; (c) generating a linker-bound
liposome by binding a linker to the hydrophilized liposome according to
need; and (d) generating a sugar-chain-modified liposome by binding a
sugar chain listed in Table 3 above to the liposome.

[0196]Preferably, in the method: the step (b) of hydrophilizing a liposome
is performed by directly or indirectly binding a low-molecular-weight
hydrophilic compound onto a lipid membrane or linker of the liposome; the
linker to be used in the step (c) is a human-derived protein (e.g., human
serum albumin); and in the step (d), under conditions where a sugar chain
is directly or indirectly bound to the liposome, a sugar-chain-modified
liposome may be generated by binding the sugar chain.

[0197]Preferably, a liposome is bound to a linker or a linker is bound to
a sugar chain using a bifunctional cross-linking agent (e.g., DTSSP) or
the like.

[0199]When used in the description, "linker" refers to a molecule that
mediates binding of a sugar chain to a liposome surface. In the
sugar-chain-modified liposome of the present invention, a sugar chain may
be bound to a liposome surface via a linker. A linker can be adequately
selected by persons skilled in the art and is preferably biocompatible
and more preferably pharmaceutically acceptable. A linker that is used in
the description can be, for example, a protein derived from a living
body, preferably a human-derived protein, more preferably a human-derived
serum protein, and further more preferably human serum albumin or bovine
serum albumin. In particular, when a human serum albumin is used, it has
been confirmed by an experiment conducted using mice that such albumin is
incorporated well into each tissue.

[0200]In the description, "cross-linking agent" refers to an agent by
which a chemical bond is formed between molecules of chain polymers in a
manner similar to that of the building of a bridge. Typically, such a
cross-linking agent refers to an agent that acts between a polymer (e.g.,
a lipid, a protein, a peptide, or a sugar chain) and another molecule
(e.g., a lipid, a protein, a peptide, or a sugar chain), so as to form a
covalent bond that links within molecules or between molecules (between
which no covalent bond is present previously). In the description, a
covalent bond may be formed between a liposome and a sugar chain with the
use of such a cross-linking agent. Alternatively, a liposome and a sugar
chain may be linked via a linker, the linker and the sugar chain may be
linked with the use of such a cross-linking agent, and the linker and the
liposome may be linked with the use of such a cross-linking agent.
Examples of such a cross-linking agent may be varied depending on targets
to be cross-linked and include, but are not limited to, aldehydes (e.g.,
glutaraldehyde), carbodiimides, and imide esters. When an amino
group-containing substance is subjected to cross-linking, an
aldehyde-containing group, such as glutaraldehyde, can be used.
Specifically, divalent reagents such as bissulfosuccinimidyl suberate,
disuccinimidyl glutarate, dithiobissuccinimidyl propionate,
disuccinimidyl suberate, 3,3'-dithiobissulfosuccinimidyl propionate,
ethylene glycol bissuccinimidyl succinate, and ethylene glycol
bissulfosuccinimidyl succinate can be used, for example.

[0201]Terms that are used in the description, "protein," "polypeptide,"
"oligopeptide," and "peptide" are used in the same sense and refer to
polymers having arbitrary lengths of amino acids. Such a polymer may be
linear or branched or in the form of a ring. "Amino acid" may be a
natural or non-natural or altered amino acid. Examples of the term may
also include those assembled to form complexes with a plurality of
polypeptide chains. Examples of the term also include natural or
artificially altered amino acid polymers. Examples of such alteration
include disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation or other arbitrary manipulation or alteration (e.g.,
formation of a conjugate with a labeling component). This definition can
also be applied to a polypeptide (containing non-natural amino acid or
the like, for example) containing 1, 2, or more analogs of amino acids, a
peptide-like compound (e.g., peptoid), and other alterations known in the
art.

[0202]In the description, it should be understood, when particularly
noted: "protein" indicates an amino acid polymer having a relatively
small molecular weight or an altered product thereof; and "peptide"
indicates an amino acid polymer having a relatively large molecular
weight or an altered product thereof. Examples of such a molecular weight
include, but are not limited to, approximately 30 kDa, preferably
approximately 20 kDa, and more preferably approximately 10 kDa.

[0203]When used in the description, "protein derived from a living body"
refers to a protein derived from an organism. Such proteins may be
derived from any organisms (e.g., arbitrary types of multicellular
organism including animals such as vertebrates and invertebrates and
plants such as monocotyledons and dicotyledons, for example). Preferably,
proteins derived from vertebrates (e.g., Hyperotreta, Hyperoartia,
Chondrichthyes, Osteichthyes, amphibians, reptiles, birds, and mammals)
and more preferably, proteins derived from mammals (e.g., Monotreme,
Marsupialia, Edentata, Dermoptera, Chiropteran, Carnivore, Insectivore,
Proboscidean, Perissodactyla, Artiodactyla, Tubulidentata, Squamata,
Sirenia, Cetacea, primates, rodents, and Lagomorpha) are used. Further
preferably, proteins derived from primates (e.g., chimpanzee, Japanese
monkey (Macaca fuscata), and human) are used. Most preferably, proteins
derived from living bodies to which administration is performed are used.

[0204]When used in the description, "human-derived serum protein" refers
to a protein that is contained in a liquid portion that remains when
human blood naturally coagulates.

[0205]When used in the description, "human serum albumin" refers to an
albumin contained in human serum, and "bovine serum albumin" refers to an
albumin contained in bovine serum.

[0206]The sugar-chain-modified liposome of the present invention may be
hydrophilized by binding a hydrophilic compound and preferably
tris(hydroxyalkyl)aminoalkane to at least one of the liposome membrane
and the linker.

[0207]When used in the description, "hydrophilization" means to bind a
hydrophilic compound to a liposome surface. Examples of a compound to be
used for hydrophilization include, a low-molecular-weight hydrophilic
compound, preferably a low-molecular-weight hydrophilic compound having
at least one OH group, and further preferably a low-molecular-weight
hydrophilic compound having at least two OH groups. Another example of
the same is a low-molecular-weight hydrophilic compound having at least
one amino group; that is, a hydrophilic compound having at least one OH
group and at least one amino group within the molecule. Such a
hydrophilic compound has a low molecular weight, so that it hardly causes
steric hindrance against sugar chains. Thus, such a hydrophilic compound
will never hinder the progress of reaction for recognition of a sugar
chain molecule, which is performed by a cell surface molecule such as a
lectin on a target cell membrane. Furthermore, in the
sugar-chain-modified liposome of the present invention, such a
hydrophilic compound does not contain a sugar chain to which a cell
surface molecule such as a lectin to be used for targeting a specific
site can bind. Examples of such a hydrophilic compound include amino
alcohols such as tris(hydroxyalkyl)aminoalkane containing
tris(hydroxymethyl)aminomethane or the like. More specific examples of
the same include tris(hydroxymethyl)aminoethane,
tris(hydroxyethyl)aminoethane, tris(hydroxypropyl)aminoethane,
tris(hydroxymethyl)aminomethane, tris(hydroxyethyl)aminomethane,
tris(hydroxypropyl)aminomethane, tris(hydroxymethyl)aminopropane,
tris(hydroxyethyl)aminopropane, and tris(hydroxypropyl)aminopropane.
Moreover, a compound prepared by introduction of an amino group into a
low-molecular-weight compound having an OH group can also be used as the
hydrophilic compound of the present invention. An example of the compound
is, but is not limited to, a compound prepared by introduction of an
amino group into a sugar chain such as cellobiose to which a cell surface
molecule (e.g., lectin) does not bind. For example, a liposome surface is
hydrophilized by applying a divalent reagent for cross-linking and
tris(hydroxymethyl)aminomethane onto lipid phosphatidyl ethanol amine of
the liposome membrane. Such a hydrophilic compound is represented by the
following general formula (1), (2), (3), or the like.

X--R1(R20H)n (formula (1))

H2N--R3--(R10H)n (formula (2))

H2N--R5(OH)n (formula (3))

Wherein R1, R3, and R5 denote C1 to C40,
preferably C1 to C20, further preferably C1 to C10
linear or branched hydrocarbon chains, R2 and R4 are absent or
denote C1 to C40, preferably C1 to C20, further
preferably C1 to C10 linear or branched hydrocarbon chains. X
denotes a reactive functional group directly binding to a liposome lipid
or a divalent reagent for cross-linking. Examples of "X" include COOH,
NH, NH2, CHO, SH, NHS-ester, maleimide, imide ester, active halogen,
EDC, pyridyl disulfide, azidophenyl, and hydrazide. N denotes a natural
number. The surface of the liposome hydrophilized using such a
hydrophilic compound is coated thinly with the hydrophilic compound.
However, the coating thickness of the hydrophilic compound is thin, so
that when a sugar chain is bound to the liposome, the reactivity of the
sugar chain or the like is not suppressed.

[0208]Hydrophilization of a liposome is performed by a conventionally
known method. For example, hydrophilization can be performed by employing
a method that involves preparing a liposome using a phospholipid to which
polyethylene glycol, polyvinylalcohol, maleic anhydride copolymer, and
the like are covalently bound (JP Patent Publication (Kokai) No.
2000-302685 A discloses that a crude dispersion of a multilayered
liposome was obtained by a method using, for example, CNDAC-containing
liposome formulation dilauroylphosphatidylcholine,
dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine,
distearoyl phosphatidylcholine; dipalmitoylphosphatidylglycerol,
distearoylphosphatidylglycerol; sphingomyelin; cholesterol; N-monomethoxy
polyethylene glycol succinyl-distearoyl phosphatidyl ethanol amine
(hereinafter, referred to as PEG2000-DSPE) in which the molecular weight
of the polyethylene glycol portion is approximately 2000; CNDAC
hydrochloride, a glucose aqueous solution, and a trehalose aqueous
solution according to the method of Bangham et al., (see J. Mol. Biol. 8,
660-668 (1964)), for example. Of these, it is particularly preferred that
a liposome surface is hydrophilized using
tris(hydroxymethyl)aminomethane. The technique of the present invention
using tris(hydroxymethyl)aminomethane is preferred because of several
reasons compared with conventional hydrophilization methods using
polyethylene glycol and the like. For example, in the present invention,
a sugar chain is bound onto a liposome and then the molecular recognition
function is used for tropism. In such a case,
tris(hydroxymethyl)aminomethane is particularly preferred since:
tris(hydroxymethyl)aminomethane that is a low-molecular-weight substance
hardly causes steric hindrance against sugar chains compared with a
conventional method using a high-molecular-weight substance such as
polyethylene glycol; and tris(hydroxymethyl)aminomethane does not hinder
the progress of a sugar chain molecule recognition reaction that is
performed by a cell surface molecule (sugar chain-recognizing protein)
such as a lectin on a target cell membrane.

[0209]Furthermore, the liposome according to the present invention has
good particle diameter distribution, component composition, and
dispersion property even after hydrophilization and is also excellent in
long-term storage stability and in vivo stability. Therefore, the
liposome is preferred when it is formulated and used. To hydrophilize the
surface of a liposome using tris(hydroxymethyl)aminomethane, for example,
a divalent reagent (e.g., bissulfosuccinimidyl suberate, disuccinimidyl
glutarate, dithiobissuccinimidyl propionate, disuccinimidyl suberate,
3,3'-dithiobissulfosuccinimidyl propionate, ethylene glycol
bissuccinimidyl succinate, and ethylene glycol bissulfosuccinimidyl
succinate) is added to a liposome solution obtained by a standard method
with the use of lipids such as dimyristoylphosphatidyl ethanol amine,
dipalmitoyl phosphatidyl ethanol amine, and distearoyl phosphatidyl
ethanol amine, so as to perform a reaction. The divalent reagent is bound
to lipids such as dipalmitoyl phosphatidyl ethanol amine on the liposome
membrane and then tris(hydroxymethyl)aminomethane is caused to react with
the other binding site of the divalent reagent, so that
tris(hydroxymethyl)aminomethane is bound to the liposome surface.

[0210]As described above, the thus hydrophilized liposome is extremely
stable in vivo. As described later, such a hydrophilic liposome has a
long half-life in vivo without binding of a sugar chain having tropism,
so that it can be appropriately used as a drug vehicle in a drug delivery
system. The present invention also encompasses a liposome hydrophilized
by treating the surface with such a low-molecular-weight compound.

[0211]In the design of the delivery vehicle based on the rolling model
discovered in the description, in vitro evaluation standard can be
determined by an experiment that is conducted for one type of lectin
(e.g., E-selectin), for example. The results of the experiment can be
listed as follows.

[0215]When used in the description, "liposome Nos." refer to numbers
corresponding to sugar-chain-modified liposomes to which sugar chains
listed in the following Table 7, Table 8, Table 9, Table 10, Table 11,
and Table 12 are bound.

[0216]A sugar-chain-modified liposome to be used in the description can
contain a sugar chain shown in Table 7 with an appropriate density for
transfer from the intestinal tract into blood, for example.

[0217]When used in the description, "modification (binding) density" is an
amount of a sugar chain that is used when a sugar-chain-modified liposome
is prepared and is represented by a density (mg sugar chain/mg lipid) of
a sugar chain that is bound per mg of the lipids of the liposome.
Regarding the binding density of the sugar-chain-modified liposome of the
present invention, it is empirically known that, although no theoretical
constraints are desired herein, the amount of a sugar chain that is used
for preparation is almost proportional to the density of the sugar chain
bound to the liposome. Therefore, in the description, unless otherwise
particularly noted, binding density is determined depending on an amount
that is used upon preparation. In vitro, for example, such binding
density can be indirectly determined using E-selectin. The tropism
(targeting) of the sugar-chain-modified liposome of the present invention
for a target delivery site can be controlled by selecting the type and
binding density of a sugar chain to be bound to a liposome. Liposome
Nos., sugar chain structures, modification (binding) densities, and
tropism in the case of oral administration (or enteral administration)
are listed in Table 7 below.

[0218]Preferably, the sugar-chain-modified liposome of the present
invention, which is appropriate for the oral administration, can be
prepared using sugar chain types and modification (binding) densities
listed in Table 7 above and combinations thereof. Once the tropism is
found to be + or ++, the similar effect can be expected, which is not
theoretically constrained, even when two or more types of sugar chain are
combined. This is because a sugar chain that is recognized to be
preferable by a lectin of target tissues or target cells is also
recognized to be preferable even when two or more types of sugar chain
are combined.

[0222]When used in the description, "modification (binding) density"
refers to an amount of a sugar chain that is used when a
sugar-chain-modified liposome is prepared. It is represented by the
density (mg sugar chain/mg lipid) of a bound sugar chain per mg of the
lipids of the liposome. Although no theoretical constraints are desired
herein, regarding the binding density of the sugar-chain-modified
liposome of the present invention, it is empirically known that the
amount of the sugar chain, which is used for preparation, is almost
proportional to the density of the sugar chain bound to the liposome.
Therefore, in the description, unless otherwise particularly noted, such
binding density is determined depending on an amount that is used upon
preparation. In vitro, for example, such binding density can be
indirectly determined using E-selectin. The tropism for a target delivery
site of the sugar-chain-modified liposome of the present invention can be
controlled by selecting the type and binding density of a sugar chain to
be bound to the liposome. Liposome numbers, sugar chain structures,
modification (binding) densities, and related tropism for tumors are
listed in Table 8 below.

[0223]When a liposome to which tris(hydroxymethyl)aminomethane is bound is
used as a standard liposome, "++" indicates that the average value of the
liposome delivered to the tumor is 1.5 to 2.5 times greater than that of
the standard liposome at 5 minutes after intravenous injection.

[0224]When a liposome to which tris(hydroxymethyl)aminomethane is bound is
used as a standard liposome, "+" indicates that the average value of the
liposome delivered to the tumor is 1.1 to 1.4 times greater than that of
the standard liposome at 5 minutes after intravenous injection.

[0225]The liposome to which tris(hydroxymethyl)aminomethane is bound has
slight tumor tropism. Thus,
Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1Cer
was used as a standard liposome.

[0226]Preferably, the sugar-chain-modified liposome of the present
invention, which is appropriate for delivery to tumors, can be prepared
using sugar chain types and modification (binding) densities listed in
Table 8 above and combinations thereof. Once the tropism is found to be +
or ++, the similar effect can be expected, although no theoretical
constraints are desired, even when two or more types of sugar chain are
combined. This is because a sugar chain that is recognized to be
preferable by a lectin of target tissues or target cells is also
recognized to be preferable even when two or more types of sugar chain
are combined.

[0228]A sugar-chain-modified liposome to be used in the description can
contain a sugar chain that is shown in Table 9, for example, at an
adequate density for delivery to inflammation sites.

[0229]When used in the description, "delivery to an inflammation site"
refers to the delivery to a region where a basic pathological process (in
which dynamic complexes are formed by cytological and histological
reactions that take place in blood vessels or tissues adjacent thereto
affected by injuries due to or abnormal stimulation with physical,
chemical, or biological action substances, for example) takes place.
Whether or not a site is an inflammation site can be confirmed by
detecting inflammatory substances (e.g., prostaglandins and
leukotrienes).

[0230]When used in the description, "modification (binding) density"
refers to an amount of a sugar chain that is used when a
sugar-chain-modified liposome is prepared. It is represented by the
density (mg sugar chain/mg lipid) of a sugar chain that is bound per mg
of the lipid of the liposome. Although it is not desired to be
theoretically constrained, regarding the binding density of the
sugar-chain-modified liposome of the present invention, it is empirically
known that the amount of a sugar chain, which is used for preparation, is
almost proportional to the density of the sugar chain bound to the
liposome. Therefore, in the description, unless otherwise particularly
noted, such binding density is determined depending on an amount that is
used upon preparation. In vitro, for example, such binding density can be
indirectly determined using E-selectin. The tropism for a target delivery
site of the sugar-chain-modified liposome of the present invention can be
controlled by selecting the type and binding density of a sugar chain to
be bound to the liposome. Liposome numbers, sugar chain structures,
modification (binding) densities, and related tropism for tumors are
listed in Table 9 below.

[0231]When a liposome to which tris(hydroxymethyl)aminomethane is bound is
used as a standard liposome, "++" indicates that the average value of the
liposome delivered to the inflammation site is 1.5 to 4.9 times greater
than that of the standard liposome at 5 minutes after intravenous
injection.

[0232]When a liposome to which tris(hydroxymethyl)aminomethane is bound is
used as a standard liposome, "+" indicates that the average value of the
liposome delivered to the inflammation site is 1.2 to 1.5 times greater
than that of the standard liposome at 5 minutes after intravenous
injection.

[0233]The liposome to which tris(hydroxymethyl)aminomethane is bound has
slight tumor tropism. Thus,
Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1Cer
was used as a standard liposome.

[0234]Preferably, the sugar-chain-modified liposome of the present
invention, which is appropriate for delivery to inflammation sites, can
be prepared using sugar chain types and modification (binding) densities
listed in Table 9 above and combinations thereof. Once the tropism is
found to be + or ++, the similar effect can be expected, which is not
theoretically constrained, even when two or more types of sugar chain are
combined. This is because a sugar chain that is recognized to be
preferable by a lectin of target tissues or target cells is also
recognized to be preferable even when two or more types of sugar chain
are combined.

[0236]A sugar-chain-modified liposome to be used in the description can
contain a sugar chain that is shown in Table 10, for example, at an
adequate density for delivery to the liver.

[0237]When used in the description, "delivery to the liver" refers to the
delivery to an area ranging from the right hypochondrium below the
diaphragm to the upper part of the epigastrium.

[0238]When used in the description, "modification (binding) density"
refers to an amount of a sugar chain that is used when a
sugar-chain-modified liposome is prepared and is represented by the
density (mg sugar chain/mg lipid) of a sugar chain that is bound per mg
of the lipid of the liposome. Although no theoretical constraints are
desired herein, regarding the binding density of the sugar-chain-modified
liposome of the present invention, it is empirically known that the
amount of a sugar chain, which is used for preparation, is almost
proportional to the density of the sugar chain bound to the liposome.
Therefore, in the description, unless otherwise particularly noted, such
binding density is determined depending on an amount that is used upon
preparation. In vitro, for example, such binding density can be
indirectly determined using E-selectin. The tropism for a target delivery
site of the sugar-chain-modified liposome of the present invention can be
controlled by selecting the type and binding density of a sugar chain to
be bound to the liposome. Liposome Nos., sugar chain structures,
modification (binding) densities, and their tropism for the liver are
listed in Table 10 below.

[0239]When a liposome to which tris(hydroxymethyl)aminomethane is bound is
used as a standard liposome, "++" indicates that the average value of the
liposome delivered to the liver is 1.5 to 2.1 times greater than that of
the standard liposome at 5 minutes after intravenous injection.

[0240]When a liposome to which tris(hydroxymethyl)aminomethane is bound is
used as a standard liposome, "+" indicates that the average value of the
liposome delivered to the liver is 1.2 to 1.5 times greater than that of
the standard liposome at 5 minutes after intravenous injection.

[0241]The liposome to which tris(hydroxymethyl)aminomethane is bound has
slight liver tropism. Thus,
Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1Cer
was used as a standard liposome.

[0242]Preferably, the sugar-chain-modified liposome of the present
invention, which is appropriate for delivery into the liver, can be
prepared using sugar chain types and modification (binding) densities
listed in Table 10 above and combinations thereof. Once the tropism is
found to be + or ++, the similar effect can be expected, which is not
theoretically constrained, even when two or more types of sugar chain are
combined. This is because a sugar chain that is recognized to be
preferable by a lectin of target tissues or target cells is also
recognized to be preferable even when two or more types of sugar chain
are combined.

[0244]A sugar-chain-modified liposome to be used in the description can
contain a sugar chain that is shown in Table 11, for example, at an
adequate density for delivery into the pancreas.

[0245]When used in the description, "delivery to the pancreas" refers to
the delivery to regions including long lobate glands (having no coat)
that extend from the flexure of the duodenum to the spleen, the flat head
part within the flexure of the duodenum, long and thin trihedral regions
across the abdominal area, and the tail part that is in contact with the
spleen.

[0246]When used in the description, "modification (binding) density"
refers to an amount of a sugar chain that is used when a
sugar-chain-modified liposome is prepared and is represented by the
density (mg sugar chain/mg lipid) of a sugar chain that is bound per mg
of the lipids of the liposome. Although no theoretical constraints are
desired herein, regarding the binding density of the sugar-chain-modified
liposome of the present invention, it is empirically known that the
amount of a sugar chain, which is used for preparation, is almost
proportional to the density of the sugar chain bound to the liposome.
Therefore, in the description, unless otherwise particularly noted, such
binding density is determined depending on an amount that is used upon
preparation. In vitro, for example, such binding density can be
indirectly determined using E-selectin. The tropism for a target delivery
site of the sugar-chain-modified liposome of the present invention can be
controlled by selecting the type and binding density of a sugar chain to
be bound to the liposome. Liposome Nos., sugar chain structures,
modification (binding) densities, and their pancreatic tropism are listed
in Table 11 below.

[0247]When a liposome (standard liposome) to which
Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1Cer
is bound instead of a sugar chain is administered via intravenous
injection, "++" indicates that the average value of the liposome
delivered into the pancreas is 2 to 4 times greater than that of the
standard liposome at 5 minutes after intravenous injection.

[0248]When a liposome (standard liposome) to which
Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1Cer
is bound instead of a sugar chain is administered via intravenous
injection, "+" indicates that the average value of the liposome delivered
into the pancreas is 1 to 2 times greater than that of the standard
liposome at 5 minutes after intravenous injection.

[0249]When a liposome to which tris(hydroxymethyl)aminomethane is bound is
used as a standard liposome, "++" indicates that the average value of the
liposome delivered into the pancreas is 1.5 to 2.2 times greater than
that of the standard liposome at 5 minutes after intravenous injection.

[0250]When a liposome to which tris(hydroxymethyl)aminomethane is bound is
used as a standard liposome, "+" indicates that the average value of the
liposome delivered into the pancreas is 1.2 to 1.5 times greater than
that of the standard liposome at 5 minutes after intravenous injection.

[0251]The liposome to which tris(hydroxymethyl)aminomethane is bound has
slight pancreatic tropism. Thus,
Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1Cer
was used as a standard liposome.

[0252]Preferably, the sugar-chain-modified liposome of the present
invention, which is appropriate for delivery into the pancreas, can be
prepared using sugar chain types and modification (binding) densities
listed in Table 11 above and combinations thereof. Once the tropism is
found to be + or ++, the similar effect can be expected, which is not
theoretically constrained, even when two or more types of sugar chain are
combined. This is because a sugar chain that is recognized to be
preferable by a lectin of target tissues or target cells is also
recognized to be preferable even when two or more types of sugar chain
are combined.

[0254]A sugar-chain-modified liposome to be used in the description can
contain a sugar chain that is shown in Table 12, for example, at an
adequate density for delivery into the brain.

[0255]When used in the description, "delivery to the brain" refers to the
delivery to regions (e.g., cerebrum, cerebellum, and medulla oblongata)
of the entire central nerve system within the cranium.

[0256]When used in the description, "modification (binding) density"
refers to an amount of a sugar chain that is used when a
sugar-chain-modified liposome is prepared and is represented by the
density (mg sugar chain/mg lipid) of a sugar chain that is bound per mg
of the lipid of the liposome. Although no theoretical constraints are
desired herein, regarding the binding density of the sugar-chain-modified
liposome of the present invention, it is empirically known that the
amount of a sugar chain, which is used for preparation, is almost
proportional to the density of the sugar chain bound to the liposome.
Therefore, in the description, unless otherwise particularly noted, such
binding density is determined depending on an amount that is used upon
preparation. In vitro, for example, such binding density can be
indirectly determined using E-selectin. The tropism for a target delivery
site of the sugar-chain-modified liposome of the present invention can be
controlled by selecting the type and binding density of a sugar chain to
be bound to the liposome. Liposome Nos., sugar chain structures,
modification (binding) densities, and their tropism for the brain are
listed in Table 12 below.

[0257]When a liposome (standard liposome) to which
Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1Cer
is bound instead of a sugar chain is administered via intravenous
injection, "++" indicates that the average value of the liposome
delivered into the brain is 3 to 7 times greater than that of the
standard liposome at 5 minutes after intravenous injection.

[0258]When a liposome (standard liposome) to which
Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1Cer
is bound instead of a sugar chain is administered via intravenous
injection, "+" indicates that the average value of the liposome delivered
into the brain is 2 to 3 times greater than that of the standard liposome
at 5 minutes after intravenous injection.

[0259]When a liposome to which tris(hydroxymethyl)aminomethane is bound is
used as a standard liposome, "++" indicates that the average value of the
liposome delivered into the brain is 1.7 to 3.7 times greater than that
of the standard liposome at 5 minutes after intravenous injection.

[0260]When a liposome to which tris(hydroxymethyl)aminomethane is bound is
used as a standard liposome, "+" indicates that the average value of the
liposome delivered into the brain is 1.1 to 1.4 times greater than that
of the standard liposome at 5 minutes after intravenous injection.

[0261]The liposome to which tris(hydroxymethyl)aminomethane is bound has
slight tropism for the brain. Thus,
Galβ1,3GalNAcβ1,4(Neu5Acα2,3)Galβ1,4Glcβ1,1Cer
was used as a standard liposome.

[0262]Preferably, the sugar-chain-modified liposome of the present
invention, which is appropriate for delivery into the brain, can be
prepared using sugar chain types and modification (binding) densities
listed in Table 12 above and combinations thereof. Once the tropism is
found to be + or ++, the similar effect can be expected, which is not
theoretically constrained, even when two or more types of sugar chain are
combined. This is because a sugar chain that is recognized to be
preferable by a lectin of target tissues or cells is also recognized to
be preferable even when two or more types of sugar chain are combined.

[0264]Sugar-chain-modified liposomes preferred in the present invention as
listed in the above Tables can be produced by the following method.
Specifically, the method comprises the steps of: (a) providing a
liposome; (b) hydrophilizing the liposome; (c) generating a linker-bound
liposome by binding a linker to the hydrophilized liposome according to
need; and (d) generating a sugar-chain-modified liposome by binding a
sugar chain shown in Table 3 above to the liposome.

[0265]Preferably, in this method, the step (b) of hydrophilizing a
liposome is performed by directly or indirectly binding a
low-molecular-weight hydrophilic compound onto the lipid membrane of the
liposome or a linker. A linker that is used in the step (c) is a protein
derived from a human. Furthermore, in the step (d), a sugar chain is
bound to the liposome so as to generate a sugar-chain-modified liposome
under conditions where the sugar chain is directly or indirectly bound to
the liposome.

[0266]A liposome and a linker are and a linker and a sugar chain are
preferably bound to each other using a bifunctional cross-linking agent
(e.g., DTSSP) or the like.

[0268]The delivery vehicle of the present invention can be used for
administering a biological factor to a subject who needs the biological
factor via oral administration. Furthermore, the delivery vehicle can
also be used for treating mammals having the disorder of the respiratory
system, circulatory system, digestive system, urinary organ
system-reproductive organ system, central nerve system, peripheral nerve
system, or the like.

[0269]The ability to control absorbance in the intestinal tract and the
specificity for the delivery to various organs of the delivery vehicle of
the present invention can also be enhanced via regulation of the
properties (e.g., depending on sugar chain types) and binding density of
the delivery vehicle. Through binding of both a sugar chain that enhances
the ability to control absorbance in the intestinal tract and a sugar
chain having tropism for a specific tissue or organ to the delivery
vehicle, a delivery vehicle having both properties of tropism for a
specific tissue or organ and ability to control absorbance in the
intestinal tract can also be prepared.

[0270]The delivery vehicle of the present invention can be easily prepared
by persons skilled in the art in view of pH, isotonicity, stability, and
the like. The delivery vehicle of the present invention can be compounded
with a pharmaceutically acceptable carrier and then administered via oral
administration in the form of solid formulation such as tablets,
capsules, fine granules, powders, or powdered drugs or liquid formulation
such as syrups, suspension agents, or solutions. The delivery vehicle can
be prescribed in a form appropriate for administration through the use of
a pharmaceutically acceptable carrier known in the art. The use of such a
carrier makes it possible to prescribe the delivery vehicle in the form
of liquid, gel, syrup, slurry, suspended matter, or the like that is
appropriate for intake by a patient.

[0271]The delivery vehicle of the present invention contains a composition
in which an active ingredient such as a drug or a biological factor is
contained in a vehicle in an effective amount (dose) for achievement of
the intended purposes. The term "effective amount (dose) for treatment"
is sufficiently recognized by persons skilled in the art and refers to an
amount of a drug, which is effective for providing intended
pharmacological results (e.g., prevention, treatment, and prevention of
recurrence). Therefore, such effective dose for treatment is an amount
sufficient for alleviating the symptoms of disease to be treated. One
useful assay for confirmation of such an effective dose (e.g., effective
dose for treatment) for a given application is to measure the degree of
recovery of a target disease. An actual dose to be administered depends
on an individual body to be treated and preferably is an amount optimized
for achieving desired effects without significant side effects.
Determination of such effective dose is sufficiently within the capacity
of persons skilled in the art.

[0272]A therapeutically effective dose, a prophylactically effective dose,
and the like and toxicity can be determined by performing standard
pharmaceutical procedures (e.g., ED50 that is a dose therapeutically
effective for 50% of a population; and LD50 that is a dose lethal to
50% of a population) for cell culture or experimental animals. The ratio
of a therapeutically effective dose to a toxic dose is a therapeutic
index that can be represented by the ratio of ED50/LD50. A drug
delivery vehicle with a small therapeutic index is preferred herein. Data
obtained via a cell culture assay and an animal experiment can be used
for formulation of the range of an amount to be used for a human. The
dose of such a compound is, preferably, within the range of a circulation
concentration including ED50 with almost no or completely no
toxicity. Such a dose is varied within the range depending on the form of
administration, which is employed herein, susceptibility of a patient,
and the route of administration. For example, such a dose is adequately
selected depending on the conditions of a patient, such as age, disease
types, cell types to be used herein, and the like.

[0273]The drug delivery vehicle of the present invention can be produced
in a manner (e.g., mixing or dissolving) similar to that known in the
art.

[0274]In the description, "instruction" describes a method or the like for
administering the sugar-chain-modified liposome, the drug delivery
vehicle for oral administration of the present invention, or the like is
described for persons who perform administration such as doctors and
patients and for persons who make diagnosis (can be patients themselves).
Such instruction contains words for instructing the procedures for
administration of the sugar-chain-modified liposome or the drug delivery
vehicle for oral administration of the present invention. Such
instruction is prepared according to the format as specified by the
supervisory authorities (e.g., Health, Labour and Welfare Ministry in
Japan and Food and Drug Administration (FDA) in the U.S.). Specifically,
such instruction is prepared according to the format as specified by the
supervisory authorities in the country in which the present invention is
implemented and the approval by the supervisory authorities is also
clearly stated therein. Such instruction is namely appended paper
(package insert) and is generally provided in the form of paper medium.
However, the form of such instruction is not limited to such form. The
instruction can also be provided in the form of electronic medium (e.g.,
homepage provided via internet (web site) or e-mail), for example.

[0275]In the description, "subject" refers to an organism to which
treatment of the present invention is applied and also refers to
"patient." Such patient or subject can be preferably a human.

[0276]The medicine of the present invention can be prepared in the form of
freeze-dried cake or aqueous solution and then stored by mixing,
according to need, a physiologically acceptable carrier, an excipient, or
a stabilizing agent (e.g., see ver. 14 or the latest version of Japanese
Pharmacopoeia and Remington's Pharmaceutical Sciences, 18th Edition,
A. R. Gennaro, ed., Mack Publishing Company, 1990) with a sugar chain
composition having a desired degree of purity.

[0277]The medicine of the present invention can be administered via oral
or parenteral administration. Alternatively, the medicine of the present
invention can be administered intravenously or subcutaneously. When
systemically-administered, a medicine to be used in the present invention
can be in the form of pharmaceutically acceptable aqueous solution
containing no pyrogen substance. Such a pharmaceutically acceptable
composition can be easily prepared by persons skilled in the art in view
of pH, isotonicity, stability, and the like. The route of administration
to be employed in the description can be oral administration, parenteral
administration (e.g., intravenous administration, intramuscular
administration, subcutaneous administration, intradermal administration,
transmucosal administration, intrarectal administration, intravaginal
administration, local administration to affected parts, and skin
administration). A product prescribed for such administration can be
provided in an arbitrary form for formulation. Examples of such form for
formulation include liquids, injections, and sustained preparations.

[0278]Examples of materials appropriate for such prescription or
pharmaceutically acceptable carriers include, but are not limited to,
anti-oxidants, preservatives, coloring agents, flavoring agents,
diluents, emulsifiers, suspending agents, solvents, fillers, extending
agents, buffers, delivery vehicles, diluents, excipients, and/or
pharmaceutical adjuvants. Typically, the medicine of the present
invention is administered in the form of composition containing an
isolated multipotent stem cell or an altered product thereof or a
derivative thereof in addition to one or more physiologically acceptable
carriers, excipients, or diluents. For example, an appropriate vehicle
can be water for injection, a physiological solution, or an artificial
cerebrospinal fluid, which can be supplemented with other general
substances for compositions for parenteral delivery.

[0280]Examples of an appropriate carrier include a neutral buffered
physiological saline solution and a physiological saline solution mixed
with serum albumin. Preferably, the generated product is prescribed as
freeze-dried agent using an appropriate excipient (e.g., sucrose).
Another standard carrier, a diluent, and an excipient can be contained if
desired. Another example of a composition contains a Tris buffer (pH7.0
to 8.5) or an acetic acid buffer (pH4.0 to 5.5). Such an example can
further contain sorbitol or an appropriate alternate thereof.

[0281]When the present invention is used for cosmetics, such cosmetics can
be prepared in compliance with the regulations defined by the authority
concerned.

[0282]The delivery vehicle or the composition of the present invention can
also be used as components of agricultural chemicals. When the delivery
vehicle or the composition of the present invention is prescribed as a
composition for an agricultural chemical, it can contain an
agriculturally acceptable carrier, an excipient, a stabilizing agent, or
the like, according to need.

[0284]The present invention can also be applied for use in the fields of
healthcare and foods. In such cases, points that should be remembered
when it is used as an oral medicine as described above should be taken
into consideration according to need. In particular, when the present
invention is applied for use as a functional food and/or health food such
as a food for specified health use, it is preferably treated according to
the manner employed for medicines. Preferably, the delivery vehicle of
the present invention can also be used as a low allergic food.

[0285]The amount of a composition that is used in the treatment method of
the present invention can be easily determined by persons skilled in the
art in view of purpose for use, target disease (e.g., type and severity),
patient's age, body weight, sex, and medical history, form or type of
cell, and the like. Frequency for performing the treatment method of the
present invention for a subject (or patient) can also be easily
determined by persons skilled in the art in view of purpose for use,
target disease (e.g., type and severity), patient's age, body weight,
sex, medical history, therapeutic process, and the like. Such frequency
for administration ranges from daily to once per several months (e.g.,
once a week to once a month), for example. It is preferable to perform
administration once a week to once a month while observing the progress.

[0286]In another aspect, the present invention provides a method for
preventing or treating a subject who needs the delivery of a drug to
desired sites. This method comprises the steps of:

A) measuring in vitro affinity of candidate delivery vehicles for
achievement of delivery to a desired site for a cell surface molecule
such as a lectin associated with the site;B) selecting a delivery vehicle
having in vitro affinity corresponding to delivery to the desired site;
andC) administering a drug required for prevention or treatment to the
subject using the selected delivery vehicle.

[0287]Alternatively, this method comprises the steps of:

A) measuring in vitro affinity of candidate delivery vehicles for
achievement of delivery to a desired site for a cell surface molecule
such as a lectin associated with the site;B) selecting a delivery vehicle
having in vitro affinity corresponding to delivery to the desired site
and analyzing the composition of the selected delivery vehicle;C)
generating the selected delivery vehicle based on the composition, which
contains a drug required for prevention or treatment; andD) administering
the selected delivery vehicle to the subject.

[0288]Examples of such delivery vehicle include, but are not limited to:

a delivery vehicle for achievement of delivery to a desired site, in which
an inhibitory concentration at approximately strong binding IC30 or less
is 10-9M or less in terms of in vitro affinity for a cell surface
molecule such as a lectin associated with a desired site;a delivery
vehicle for achievement of delivery to a desired site, in which an
inhibitory concentration at approximately weak binding IC31 or more is
10-9M or more in terms of in vitro affinity for a cell surface
molecule such as a lectin associated with a desired site;a delivery
vehicle for achievement of delivery to a desired site, in which an
inhibitory concentration at approximately strong binding IC30 or less is
10-9M or less, and an inhibitory concentration at approximately weak
binding IC31 or more is 10-9M or more in terms of in vitro affinity
for a cell surface molecule such as a lectin associated with a desired
site; anda delivery vehicle for achievement of delivery to a desired
site, which satisfies at least one condition selected from the group
consisting of a condition in which the inhibitory concentration at IC40
is 10-9M or more, a condition in which the inhibitory concentration
at IC50 is 10-9M or more, and a condition in which the inhibitory
concentration at IC40 is 10-9M or more, and satisfies at least one
condition selected from the group consisting of a condition in which the
inhibitory concentration at IC30 is 10-9M or less, a condition in
which the inhibitory concentration at IC20 is 10-9M or less, and a
condition in which the inhibitory concentration at IC10 is 10-9M in
terms of in vitro affinity for a cell surface molecule such as a lectin
associated with a desired site. Here, IC can be measured using affinity
for E-selectin, but the example is not limited thereto. It is understood
that these delivery vehicles are also encompassed within the scope of the
present invention. In this case, since E-selectin closely correlates with
at least inflammation sites and cancer sites, the delivery vehicles can
be used for delivery to the inflammation sites and cancer sites.

[0289]Specific examples of sugar-chain-modified liposomes that satisfy the
above conditions can be listed as follows.

[0290]Preferably, a delivery vehicle can be a liposome (e.g.,
sugar-chain-modified liposome).

[0291]Therefore, the present invention also provides a pharmaceutical
composition containing a drug that is used for prevention or treatment
and the delivery vehicle of the present invention or a delivery vehicle
that is produced by the method for producing a delivery vehicle of the
present invention.

(Alteration)

[0292]In the description, a delivery vehicle that is actually measured can
be altered by substitution, if desired. In the case of a sugar chain, the
specificity of the sugar chain can be altered by introducing a methyl
group for substitution of a hydroxy group of the sugar chain, for
example. The affinity of a product prepared by such alteration can be
measured by in vitro screening according to the rolling model of the
present invention.

[0293]In the description, unless otherwise particularly noted,
"substitution" refers to substitution of 1, 2 or more (or several)
hydrogen atoms in an organic compound or a substituent with other atoms
or atomic groups. Substitution with a monovalent substituent can also be
performed by the removal of one hydrogen atom. Furthermore, substitution
with a divalent substituent can also be performed by the removal of two
hydrogen atoms.

[0294]In the description, unless otherwise particularly noted,
"substitution" refers to substitution of 1, 2, or more hydrogen atoms in
an organic compound or a substituent with other atoms or atomic groups.
Substitution with a monovalent substituent can also be performed by the
removal of one hydrogen atom. Furthermore, substitution with a divalent
substituent can also be performed by the removal of two hydrogen atoms.

[0295]Examples of a substituent to be used in the present invention
include, but are not limited to, alkyl, cycloalkyl, alkenyl,
cycloalkenyl, alkynyl, alkoxy, carbocyclic group, heterocyclic group,
halogen, hydroxy, thiol, cyano, nitro, amino, carboxy, carbamoyl, acyl,
acylamino, thio carboxy, amido, substituted carbonyl, substituted thio
carbonyl, substituted sulfonyl, and substituted sulfinyl. Such
substituent can be adequately used when amino acids are designed in the
present invention.

[0296]Preferably, a plurality of such substituents are present, they can
be each independently a hydrogen atom or alkyl. However, it is not
required that all of these plural number of substituents are hydrogen
atoms. More preferably, when a plurality of such substituents are
independently present, they can be each independently selected from the
group consisting of hydrogen and C1 to C6 alkyl. All of these
substituents may have substituents other than hydrogen. Preferably, these
substituents can have at least one hydrogen, more preferably, 2 to n
(here "n" denotes the number of substituent) hydrogens. Preferably, the
number of hydrogen may be greater than that of substituents other than
hydrogen. This is because a small substituent or a substituent with
polarity can cause damage to the effects of the present invention
(particularly, interaction with an aldehyde group). Therefore,
substituents other than hydrogen can be preferably C1 to C6 alkyl, C1 to
C5 alkyl, C1 to C4 alkyl, C1 to C3 alkyl, C1 to C2 alkyl, methyl, and the
like. Furthermore, such a small substituent can also enhance the effects
of the present invention, so that possession of a small substituent is
also preferred herein.

[0297]In the description, "C1, C2 . . . , and Cn" denote the number of
carbon. Therefore, C1 is used to denote a substituent having one carbon.

[0298]In the description, "protection reaction" refers to a reaction by
which a protecting group such as Boc is added to a functional group
desired to be protected. When a functional group is protected with a
protecting group, the reaction of functional groups with higher
reactivity can be suppressed and only the functional groups with lower
reactivity can be caused to react. Such a protection reaction can be
performed by dehydration reaction, for example.

[0299]In the description, "deprotection reaction" refers to a reaction by
which a protecting group such as Boc is deprotected. An example of such a
deprotection reaction is a reduction reaction using Pd/C. Deprotection
reaction can be performed by hydrolysis, for example.

[0300]In the description, examples of typical "protecting group" include
fluorenylmethoxycarbonyl (Fmoc) group, acetyl group, benzyl group,
benzoyl group, t-butoxy carbonyl group, t-butyl dimethyl group, silyl
group, trimethyl silyl ethyl group, N-phthalimidyl group, trimethyl silyl
ethyloxy carbonyl group, 2-nitro-4,5-dimethoxy benzyl group,
2-nitro-4,5-dimethoxy benzyloxycarbonyl group, and carbamate group. Such
a protecting group can be used to protect a reactive functional group
such as an amino group or a carboxyl group, for example. Various
protecting groups can be separately used according to conditions or
purposes for reaction. An acetyl group, a benzyl group, a silyl group, or
a derivative thereof can be used as a protecting group for a hydroxy
group. In addition to an acetyl group, a benzyloxycarbonyl group, a
t-butoxy carbonyl group, or a derivative thereof can be used as a
protecting group for an amino group. As a protecting group for an amino
oxy group or an N-alkylamino oxy group, a trimethyl silyl ethyloxy
carbonyl group, a 2-nitro-4,5-dimethoxy benzyloxycarbonyl group, or a
derivative thereof is preferred.

[0301]In each method of the present invention, a product to be generated
can be isolated by removing foreign substances (e.g., unreacted raw
materials, by-products, and solvents) from the reaction solution by a
method that is generally used in the art (e.g., extraction, distillation,
washing, condensation, precipitation, filtration, and drying) and then
performing a combination of post-treatment methods that are generally
used in the art (e.g., adsorption, elution, distillation, precipitation,
deposition, and chromatography).

[0302]In the present invention, it is understood that an addition reaction
of a sugar chain proceeds in principle as long as contact takes place.
Preferably, for example, it is understood that such reaction proceeds at
25° C. to 80° C. Examples of the upper limit of appropriate
temperatures include, but are not limited to, 80° C., 70°
C., 60° C., 50° C., 42° C., and 40° C. Such a
temperature varies depending on the type of a protein. The upper limit of
a protein that is easily heat-denatured can be 37° C., for
example. The lower limit of appropriate temperatures can be 25°
C., 30° C., 32° C., 37° C., or the like. The lower
limit of appropriate temperatures can be adequately determined by persons
skilled in the art in connection with reaction speed and in view of
necessary time.

[0303]Reaction time (time required for reaction) can also be adequately
determined by persons skilled in the art based on the information
contained in the description. Such reaction time ranges from 6 hours to 5
days, for example, but the example is not limited thereto.

[0304]Examples of the lower limit of such reaction time include, but are
not limited to, several hours (e.g., 1 hour, 2 hours, 3 hours, 4 hours, 5
hours, and 6 hours), 1 day, and several days (2 to 3 days). Persons
skilled in the art can adequately determine the reaction time by taking
reaction speed, efficiency, and the like into consideration based on the
information contained in the description. Examples of the upper limit of
the reaction time include, but are not limited to, several days (2 to 3
days), 5 days, 6 days, and 10 days. It is desired to determine the upper
limit of the reaction time so that the thus produced glycoprotein is not
degraded or denatured.

(Method for Producing Sugar-Chain-Modified Liposome)

[0305]In another aspect, the present invention provides a method for
producing a sugar-chain-modified liposome. This method comprises the
steps of: (a) providing a liposome; (b) hydrophilizing the liposome; (c)
binding a linker to the hydrophilized liposome according to need, so as
to generate a linker-bound liposome; and (d) binding a sugar chain to the
liposome so as to generate a sugar-chain-modified liposome. Preferably,
in this method, the step (b) of hydrophilizing a liposome is performed by
binding directly or indirectly a low-molecular-weight hydrophilic
compound (e.g., tris(hydroxyalkyl)aminoalkane) onto the lipid membrane of
the liposome or the linker, the linker that is used in the step (c) is a
human-derived protein (e.g., human serum albumin), and a
sugar-chain-modified liposome is generated in the step (d) of binding a
sugar chain under conditions where the sugar chain is directly or
indirectly bound to the liposome.

[0306]In another aspect, the present invention provides a method for
producing a sugar-chain-modified liposome for delivery of a drug to a
target delivery site. This method comprises the steps of: (a) providing
sugar-chain-modified liposomes varying in sugar chain density for
achievement of delivery to a target delivery site; (b) determining the
sugar chain density of a sugar-chain-modified liposome for achievement of
optimum delivery to the delivery site; and (c) incorporating the drug
into the thus determined optimum sugar-chain-modified liposome so as to
generate a drug-containing liposome.

(Production of Liposome)

[0307]A liposome itself can be produced according to a known method.
Examples of such a method include a thin film method, a reverse phase
evaporation method, an ethanol injection method, and a
dehydration-rehydration method.

[0308]Moreover, the particle diameter of a liposome can also be regulated
using an ultrasonic irradiation method, an extrusion method, a French
press method, a homogenization method, or the like. A method for
producing the liposome itself of the present invention is more
specifically described as follows. For example, first, a lipid(s)
compounded with phosphatidyl cholines, cholesterol, phosphatidyl ethanol
amines, phosphatidic acids, gangliosides, glycolipids, or phosphatidyl
glycerols as an ingredient is mixed with surfactant sodium cholate to
prepare a mixed micelle. In particular, compounding with phosphatidic
acids or long-chain alkyl phosphates such as dicetylphosphate is
essential for negatively charging the liposome. Compounding with
phosphatidyl ethanol amines is essential as a hydrophilic reaction site.
Compounding with gangliosides or glycolipids or phosphatidyl glycerols is
essential as a binding site of a linker. At least one type of lipid
selected from the group consisting of gangliosides, glycolipids,
phosphatidyl glycerols, sphingomyelins, and cholesterols assembles in the
liposome and then functions as a foothold (raft) for binding the linker.
The liposome of the present invention is further stabilized by the
formation of such raft to which a protein can bind. Specifically, an
example of the liposome of the present invention is a liposome in which a
raft (for binding with a linker) of at least one type of lipid selected
from the group consisting of ganglioside, glycolipid, phosphatidyl
glycerols, sphingomyelins, and cholesterols. The thus obtained mixed
micelle is subjected to ultrafiltration, so that a liposome is prepared.
A general liposome can be used in the present invention and the surface
of such a liposome is desirably hydrophilized in advance. After a
liposome is prepared as described above, the liposome surface is
hyrophilized.

[0309]The present invention further encompasses a liposome itself, to
which a hydrophilized sugar chain (hydrophilized using the above
hydrophilic compound) is not bound. Such a hydrophilized liposome has
advantages such that it has enhanced stability of its own or the
recognition ability of a sugar chain is improved when the sugar chain is
bound. The liposome of the present invention is a liposome that contains
a constitutive lipid of the liposome that is at least one or more types
of lipid (mole percentage: 0% to 30%) selected from the group consisting
of phosphatidyl cholines (mole percentage: 0% to 70%), phosphatidyl
ethanol amines (mole percentage: 0% to 30%), phosphatidic acids,
long-chain alkyl phosphate, and dicetylphosphates, at least one or more
types of lipid (mole percentage: 0% to 40%) selected from the group
consisting of gangliosides, glycolipids, phosphatidyl glycerols, and
sphingomyelins, and cholesterols (mole percentage: 0% to 70%), for
example.

[0310]The present invention further encompasses a method for
hydrophilizing a liposome by further binding the above hydrophilic
compound to a liposome so as to hydrophilize the liposome. Moreover, the
present invention encompasses a hydrophilized liposome having no sugar
chain bound thereto. The targeting liposome or the intestinal tract
absorbable liposome of the present invention can be produced by binding a
sugar chain to a liposome having no sugar chain bound thereto.

(Synthesis of Sugar Chain)

[0311]A sugar chain that can be used for the sugar-chain-modified liposome
of the present invention can be synthesized by a general method for
synthesizing a sugar chain. Examples of such method include (1) a method
that involves chemical synthesis, (2) a fermentation method using gene
recombinant cells or microorganisms, (3) a synthesis method using
glycosidase, (4) and a synthesis method using glycosyltransferase. (see
WO2002/081723, JP Patent Publication (Kokai) No. 9-31095 A (1997), JP
Patent Publication (Kokai) No. 11-42096 A (1999), JP Patent Publication
(Kokai) No. 2004-180676 A, and Kenichi Hatanaka, Shinichiro Nishimura,
Tatsuro Ouchi, and Kazukiyo Kobayashi (1997) Glycoscience and
Glycotechnology (To-shisu no kagaku to kogyo), Kodansha Scientific Ltd.,
Tokyo, for example). A sugar chain that is used in the
sugar-chain-modified liposome of the present invention may be a sugar
chain that is synthesized by the above method or a commercially available
sugar chain.

(Binding of Sugar Chain to Liposome)

[0312]In the present invention, any one of the above sugar chains may be
directly bound or indirectly bound via a linker to a liposome prepared as
described above. At this time, the number of types of sugar chain to be
bound to a liposome is not limited to one type and a plurality of sugar
chains may also be bound. Such a plurality of sugar chains may have
activity of binding to different cell surface molecules such as lectins
that commonly exist on the cell surfaces of the same tissues or organs.
Alternatively, such a plurality of sugar chains may have activity of
binding to different cell surface molecules such as lectins that exist on
the cell surfaces of the different tissues or organs. Through the
selection of the former (plurality of) sugar chains, targeting to
specific target tissues or organs can be ensured. Through the selection
of the latter (plurality of) sugar chains, a single type of liposome can
be caused to target to a plurality of targets so that a multi-purpose
targeting liposome can be obtained.

[0313]In addition, to bind a sugar chain to a liposome, a linker and/or a
sugar chain is mixed when a liposome is produced and then the sugar chain
can be caused to bind to the surface of the liposome while producing the
liposome. It is desired that a liposome, a linker, and a sugar chain are
separately prepared in advance and the linker and/or the sugar chain is
bound to the produced liposome (production is completed). This is because
the density of a sugar chain to be bound can be controlled by causing a
linker and/or a sugar chain to bind to the liposome. Direct binding of a
sugar chain to a liposome can be performed by a method described below.

[0314]A liposome is produced by mixing a sugar chain as a glycolipid.
Alternatively, a sugar chain is bound to a phospholipid of the thus
produced liposome while controlling the sugar chain density. When a sugar
chain is bound using a linker, a living body-derived protein, and
particularly a human-derived protein is preferably used as a linker.
Examples of such a living body-derived protein include, but are not
limited to proteins existing in blood, such as albumin and other
physiologically active substances existing in a living body. Examples of
such proteins include serum albumins of animals such as human serum
albumin (HSA) and bovine serum albumin (BSA). Particularly, when human
serum albumin is used, it has been confirmed in experiments using mice
that the amount of HSA incorporated in each tissue is high. The liposome
of the present invention is very stable so that it makes it possible to
perform posttreatment such as binding of a protein, binding of a linker,
or binding of a sugar chain after liposome formation. Therefore, when a
large amount of a liposome is produced, different proteins are bound or a
linker or a sugar chain is bound according to purposes, so that various
liposomes can be produced for different purposes.

[0315]A sugar chain is bound indirectly via a linker or directly to the
constituent lipids of the liposome of the present invention. The liposome
of the present invention has a glyoconjugate ligand such as a glycolipid
or a glycoprotein and is hydrophilized with the use of a
low-molecular-weight compound.

[0316]Furthermore, when the targeting liposome of the present invention is
used as a medicine as described later, the liposome is required to
contain a compound with medicinal effects. Such a compound having
medicinal effects may be encapsulated in a liposome or bound to the
liposome surface. A protein having medicinal effects may be used as a
linker. In this case, a protein plays both roles of a linker for binding
a sugar chain to a liposome and a protein having medicinal effects. An
example of such a protein having medicinal effects is a physiologically
active protein.

[0317]Binding of a sugar chain to a liposome via a linker may be performed
by a method described below.

[0318]First, a protein is bound to the surface of a liposome. The liposome
is treated with an oxidant such as NaIO4,
Pb(O2CCH3)4, and NaBiO3, so as to oxidize liposome
ganglioside existing on the liposome membrane surface. Next, the linker
and the ganglioside on the liposome membrane surface are bound by a
reductive amination reaction using a reagent such as NaBH3CN and
NaBH4. The linker is also preferably hydrophilized. For this
purpose, a compound having a hydroxy group is bound to the linker
protein. For example, with the use of a divalent reagent such as
bissulfosuccinimidyl suberate, disuccinimidyl glutarate,
dithiobissuccinimidyl propionate, disuccinimidyl suberate,
3,3'-dithiobissulfosuccinimidyl propionate, ethylene glycol
bissuccinimidyl succinate, and ethylene glycol bissulfosuccinimidyl
succinate, the above compound to be used for hydrophilization, such as
tris(hydroxymethyl)aminomethane may be bound to the linker on the
liposome.

[0319]This process is more specifically described below. First, one end of
a cross-linking divalent reagent is bound to all the amino groups of the
linker. Next, sugar chain glycosylamine compounds are prepared by
performing glycosylamination (reaction) of the reducing terminus of each
type of sugar chain. The amino group of each sugar chain is bound to the
other unreacted terminus (corresponding to a part of) of the above-bound
cross-linking divalent reagent on the liposome. A covalent bond between
the sugar chain and/or hydrophilic compound and the liposome or a
covalent bond between the sugar chain and/or hydrophilic compound and the
linker may be cleaved when the liposome is incorporated into cells. For
example, when a linker and a sugar chain are covalently bound via a
disulfide bond, the sugar chain is cleaved as a result of intracellular
reduction. As a result of cleavage of the sugar chain, the liposome
surface becomes hydrophobic and binds to biomembrane, so that membrane
stability is disturbed and a drug contained in the liposome is released.

[0320]Next, hydrophilization treatment is performed using most of the thus
obtained unreacted termini of the divalent reagent (which have remained
unreacted; that is, no sugar chain is bound thereto) which have remained
on the surfaces of proteins on the sugar chain-bound liposome membrane
surfaces. Specifically, a binding reaction is performed between unreacted
termini of the divalent reagent binding to the proteins on the liposome
and the above compound to be used for hydrophilization, such as
tris(hydroxymethyl)aminomethane. Thus, the entire liposome surface is
hydrophilized. Hydrophilization of a liposome surface and a linker
improves transferability to various types of tissue, retentivity in
blood, and transferability to various types of tissue. This may be caused
by that portions other than sugar chains in each tissue or the like are
observed as if they are in vivo water contents as a result of
hydrophilization of the liposome surface and the linker surface, so that
tissues and the like other than the target are not recognized and only
the sugar chain is recognized by a cell surface molecule such as a lectin
(sugar chain-recognizing protein) of the target tissue.

[0321]Subsequently, the sugar chain is bound to the linker on the
liposome. For this, the reducing termini of saccharides composing the
sugar chain are glycosylaminated using an ammonium salt such as
NH4HCO3 and NH2COONH4. Next, with the use of a
divalent reagent such as bissulfosuccinimidyl suberate, disuccinimidyl
glutarate, dithiobissuccinimidyl propionate, disuccinimidyl suberate,
3,3'-dithiobissulfosuccinimidyl propionate, ethylene glycol
bissuccinimidyl succinate, and ethylene glycol bissulfosuccinimidyl
succinate, the linker bound onto the liposome membrane surface is bound
to the above glycosylaminated saccharide, so that a liposome complex as
shown in FIG. 1 is obtained. In addition, these sugar chains are
commercially available.

[0322]The particle diameter of the liposome or that of the liposome bound
with a sugar chain or the like of the present invention ranges from 30 nm
to 500 nm, and preferably 50 nm to 350 nm. Furthermore, desirably the
liposome of the present invention is negatively charged. If the liposome
is negatively charged, its interaction with negatively charged cells in
vivo can be avoided. The zeta potential of the liposome surface of the
present invention ranges from -50 mV to 10 mV, preferably -40 mV to 0 mV,
and further preferably -30 mV to -10 mV at 37° C. in a
physiological saline solution.

[0324]When the above drug is contained in the liposome of the present
invention, the liposome can be used for treating diseases such as cancer
and inflammation. Here, "cancer" includes diseases due to all neoplasms
such as tumor and leukemia. When such drug is contained in the
sugar-chain-modified liposome of the present invention and then the
liposome is administered, the drug is accumulated in a cancer or an
inflammation site at a higher level than that in a case in which the drug
alone is administered. Specifically, the drug contained in the liposome
of the present invention can be accumulated at a level 2 or more times,
preferably 5 or more times, further preferably 10 or more times, and
particularly preferably 50 or more times greater than that in the case in
which the drug alone is administered.

[0325]Furthermore, a compound having medicinal effects may be encapsulated
in a liposome or bound to the surface of a liposome. For example, a
protein can be bound to such a surface by the same method as the above
method for binding a linker. Other compounds can also be bound by a known
method using functional groups of the compounds. Moreover, encapsulation
into a liposome can be performed by the following method. A known method
may be used for encapsulating a drug and the like into a liposome. For
example, a liposome is formed using a solution containing a drug and the
like and a lipid(s) including phosphatidyl cholines, phosphatidyl ethanol
amines, phosphatidic acids or long-chain alkyl phosphates, gangliosides,
glycolipids or phosphatidyl glycerols and cholesterols. A drug and the
like are then encapsulated into the liposome.

[0326]Therefore, a liposome formulation is obtained by encapsulating a
drug or a gene that can be used for treatment or diagnosis into the
liposome of the present invention. The liposome formulation has
selectively-controlled transferability to cancer tissues, inflammatory
tissues, and various types of tissue. The liposome formulation can enable
enhanced effects of a therapeutic drug or a diagnostic agent via its
accumulation in a concentrated manner in target cells and tissues,
alleviated side effects due to reduced incorporation of the drug in other
cells and tissues, or the like.

[0327]Moreover, when the drug delivery vehicle for intravenous injection
and oral administration of the present invention is used for diagnosis, a
labeling compound such as a fluorescent pigment or a radioactive compound
is encapsulated in or bound to a liposome. The labeling compound-bound
liposome binds to an affected part, the labeling compound is incorporated
into the cells of the affected part, and then disease can be detected
and/or diagnosed using the presence of the labeling compound as an
indicator.

[0328]When the present invention is applied for use in diagnosis, for
example, it can be applied for DNA probe diagnostic agents, X-ray
contrast materials, radioactive reagents, radioactive contrast materials,
radioactive diagnostic agents, fluorescent reagents, fluorescent contrast
materials, fluorescent diagnostic agents, contrast materials for CT,
contrast materials for PET, contrast materials for SPECT, contrast
materials for MRI, diagnostic agents for AIDS, reagents for hematological
tests, reagents for functional tests, reagents for microbial tests,
molecular imaging, in vivo imaging, fluorescent imaging, luminescence
imaging, cell sorters, PET and SPECT, and the like. Examples of a
research reagent include reagents that are used in DNA recombination
technology, an immunoassay, a hybridization method, and an enzyme assay.

[0329]For example, as a result of the present invention; that is, as
demonstrated in Example 9, Example 10, Example 22A, and Example 22B, the
sugar-chain-modified liposome highly effectively accumulates and delivers
drugs, fluorescent substances, radioactively labeled substances, or the
like in parts affected by diseases and various organs based on active
targeting using the functions of the sugar chains as ligands. Therefore,
the sugar-chain-modified liposome of the present invention makes it
possible to visualize the accumulation in target tissues such as tumors.
Thus, according to the present invention, in addition to the use of the
liposome as a delivery vehicle for delivering a drug for treatment, a
delivery vehicle to be used as a reagent for research or a diagnostic
agent is also provided.

[0330]When the present invention is applied for use in diagnosis, for
example, it can be applied for DNA probe diagnostic agents, X-ray
contrast materials, radioactive diagnostic agents, fluorescent diagnostic
agents, contrast materials for CT, contrast materials for PET, contrast
materials for SPECT, contrast materials for MRI, diagnostic agents for
AIDS, reagents for hematological tests, reagents for functional tests,
reagents for microbial tests, molecular imaging, in vivo imaging,
fluorescent imaging, luminescence imaging, cell sorters, PET and SPECT,
and the like. Examples of a research reagent include reagents that are
used in DNA recombination technology, an immunoassay, a hybridization
method, and an enzyme assay.

(Beauty and Makeup)

[0331]In another embodiment, an object of the present invention can be
therapy, treatment, or improvement for beauty. Examples of such an object
include not only beauty and makeup treatment performed for purely healthy
subjects, but also beauty treatment performed for postsurgical or
posttraumatic deformity or for congenital deformity. For example, the
present invention can be used for tissue augmentation of breast (breast
augmentation), tissue augmentation for the recesses of cheek or upper and
lower eyelids, and tissue augmentation for facial hemiatrophy, tissue
atrophy after facial paralysis, funnel chest, and the like. Furthermore,
the present invention can also be used for nose job, reduction
rhinoplasty, genioplasty (tissue augmentation), metopoplasty (tissue
augmentation), and otoplasty (plasty for auricular cartilage) that is
performed for malformed ears-malformation such asmicrotia, but the
examples are not limited thereto.

[0332]When the present invention is used in the fields of beauty and
makeup, such composition can further contain a pharmaceutically
acceptable carrier and the like. Examples of such a pharmaceutically
acceptable carrier that is contained in the medicine of the present
invention include arbitrary substances known in the art.

[0335]Examples of an appropriate carrier include a neutral buffered
physiological saline solution and a physiological saline solution mixed
with serum albumin. Preferably, the generated product is prescribed as a
freeze-dried agent using an appropriate excipient (e.g., sucrose).
Another standard carrier, a diluent, and an excipient can be contained if
desired. Another example of a composition contains a Tris buffer (pH7.0
to 8.5) or an acetic acid buffer (pH4.0 to 5.5). Such an example can
further contain sorbitol or an appropriate alternate thereof.

(Healthcare and-Food)

[0336]The present invention can also be applied in the fields of
healthcare and foods. In such cases, points that should be remembered
when it is applied for oral medicines as described above should be taken
into consideration according to need. In particular, when the present
invention is used for functional foods and/or health foods such as a food
for specified health use, it is preferably treated according to the
manner employed for medicines. Preferably, the sugar-chain-modified
liposome of the present invention into which a functional food, a
nutritional supplement, or a health supplement is encapsulated or bound
can be used as a food composition. Such a functional food, nutritional
supplement, or health supplement that can be used in the present
invention is not limited. Examples of such food include any foods as long
as they are designed, processed, and then converted so that the food
functions are effectively expressed after intake.

[0337]Examples of such a functional food, nutritional supplement, or
health supplement that can be used in the present invention include
ginkgo leaves, echinacea, saw palmetto, ST John's Wort, valerian, black
cohosh, milk thistle, evening primrose, grape seed extracts, Vaccinium
myrtillus, feverfew, Angelica root, soybean, French maritime pine bark,
garlic, Asian ginseng, tea, ginger, Agaricus, mesimakobu, purple ipe,
AHCC, yeast beta-glucan, Grifola frondosa, propolis, brewer's yeast,
cereals, Japanese plum, chlorella, young leaves of barley, green juice,
vitamins, collagen, glucosamine, mulberry leaves, Rooibos tea, amino
acid, royal jelly, shiitake mushroom (mycelium) extracts, spirulina,
Denshichi ginseng, cress, plant fermentation foods, DHA, EPA, ARA,
Laminaria japonica (kombu), cabbage, aloe, megusurinoki (paperbark
maple), hop, oyster extracts, pycnogenol, and sesame. They may be
directly contained in liposomes or treated products such as extracts or
the like obtained therefrom may also be contained. A food composition
containing a liposome is orally ingested. A liposome to be used herein
may be a liposome to which no sugar chain is bound or a liposome to which
a sugar chain for enhancing intraintestinal absorption or a sugar chain
targeting a specific tissue or organ is bound. When the liposome of the
present invention is administered as a food composition, the liposome may
be processed into a food such as a liquid beverage, a gelled food, or a
solid food. The liposome may also be processed into tablets, granules, or
the like. The food composition of the present invention can be used as a
functional food, a nutritional supplement, or a health supplement
according to the types of food in which the liposome is contained. For
example, such foods may contain vitamins, minerals, amino acids, and
carbohydrates.

[0338]For example a liposome containing DHA can be used as a functional
food, a nutritional supplement, or a health supplement effective for mild
senile dementia or memory improvement.

(Production Apparatus)

[0339]In another aspect, the present invention provides an apparatus for
producing a delivery vehicle for achieving delivery to a desired site.
The apparatus is provided with:

A) a means for measuring in vitro affinity of candidate delivery vehicles
for a cell surface molecule such as a lectin associated with the site;
andB) a means for selecting a delivery vehicle having in vitro affinity
corresponding to delivery to the desired site.

[0340]Alternatively, the present invention provides an apparatus for
producing a delivery vehicle for achieving delivery to a desired site.
This apparatus is provided with:

A) a means for measuring in vitro affinity of candidate delivery vehicles
for a cell surface molecule such as a lectin associated with the site;B)
a means for selecting a delivery vehicle having in vitro affinity
corresponding to delivery to the desired site.C) a means for analyzing
the composition of the selected delivery vehicle; andD) a means for
generating the selected delivery vehicle based on the composition.

(Use)

[0341]In another aspect, the present invention provides the use of in
vitro affinity for a cell surface molecule such as a lectin associated
with a desired site, for production of a delivery vehicle for achieving
delivery to the desired site. Such in vitro affinity can be used for
various purposes. For example, such in vitro affinity can be used not
only for therapeutic drugs, but also for diagnostic agents (e.g., a
contrast material for MRI), research reagents (e.g., a fluorescent
probe), cosmetics, and functional foods. Furthermore, such in vitro
affinity can also be used not only for products in the agricultural,
medical, and pharmaceutical fields (including pharmaceutical products,
cosmetic-agricultural chemical-foods), but also for reagents for assay.
Such concept has been absent before disclosure of the present invention.
Hence, the significance of such concept is of great significance.

[0342]References such as scientific publications, patents, and patent
applications cited herein are incorporated herein by reference in their
entirety to a degree such that each reference is specifically described.

[0343]As described above, the present invention is illustrated using the
preferred embodiments of the present invention. However, the present
invention should not be construed in a manner limited to these
embodiments. It is understood that the scope of the present invention
should be construed based only on the claims. It is understood that
persons skilled in the art can implement the present invention within a
scope equivalent to that based on specific descriptions of the preferred
embodiments of the present invention and technical commonsense. It is
understood that all patents, patent applications, and publications cited
herein are incorporated herein by reference in their entirety to a degree
such that the contents thereof are specifically described herein.

EXAMPLES

[0344]The constitution of the present invention will be further described
below specifically with reference to examples. However, the present
invention is not limited by the following examples. Reagents used in the
following examples were commercially available reagents, unless otherwise
particularly noted.

Example 1

Preparation of Liposome

[0345]A liposome was prepared by the techniques of the previous report
(Yamazaki, N., Kodama, M. and Gabius, H.-J. (1994) Methods Enzymol. 242,
56-65) using improved cholic acid dialysis. Specifically,
dipalmitoylphosphatidylcholine, cholesterol, dicetylphosphate,
ganglioside, and dipalmitoylphosphatidylethanol amine were mixed at a
molar ratio of 35:40:5:15:5 so that the total amount of lipid was 45.6
mg. 46.9 mg of sodium cholate was added to the mixture and then the
resultant was dissolved in 3 ml of a chloroform/methanol solution. The
solution was evaporated and then the precipitate was dried in vacuum,
thereby obtaining a lipid membrane. The thus obtained lipid membrane was
suspended in 3 ml of a TAPS buffer solution (pH 8.4) and then the
resultant was ultrasonicated, so that a transparent micelle suspension
was obtained. Furthermore, the micelle suspension was subjected to
ultrafiltration using a PM10 membrane (Amicon Co., U.S.A.) and a
phosphate buffer (pH 7.2, Phosphate Buffered Saline (PBS):
Na2HPO4 (25.55 g)/KH2PO4 (2.72 g)/NaN3 (0.8
g)/NaCl (35.4 g)). Thus, 10 ml of homogeneous liposome (average particle
diameter: 100 nm) was prepared.

Example 2

Hydrophilization of Liposome Lipid Membrane Surface

[0346]10 ml of the liposome solution prepared in Example 1 was subjected
to ultrafiltration using an XM300 membrane (Amicon Co., U.S.A.) and a CBS
buffer solution (pH 8.5) and the pH of the solution was adjusted to pH
8.5. Next, 10 ml of a cross-linking reagent
bis(sulfosuccinimidyl)suberate (BS3; Pierce Co., U.S.A.) was added,
followed by 2 hours of agitation at 25° C. Subsequently, the
solution was further agitated overnight at 7° C. so as to complete
the chemical binding reaction between lipid
dipalmitoylphosphatidylethanol amine on the liposome membrane and BS3.
The liposome solution was then subjected to ultrafiltration using an
XM300 membrane and a CBS buffer solution (pH 8.5). Next, 40 mg of
tris(hydroxymethyl)aminomethane dissolved in 1 ml of a CBS buffer
solution (pH 8.5) was added to 10 ml of the liposome solution, followed
by 2 hours of agitation at 25° C. The solution was then agitated
overnight at 7° C., so as to complete the chemical binding
reaction between BS3 bound to the lipids on the liposome membrane and
tris(hydroxymethyl)aminomethane. Thus, the hydroxy group of
tris(hydroxymethyl)aminomethane was coordinated on the lipid
dipalmitoylphosphatidylethanol amine of the liposome membrane, so that
the liposome membrane surface was hydrated and hydrophilized.

Example 3

Binding of Human Serum Albumin (HSA) onto Liposome Membrane Surface

[0347]Human serum albumin (HSA) was bound onto a liposome membrane surface
according to the technique of the previous report (Yamazaki, N., Kodama,
M. and Gabius, H.-J. (1994) Methods Enzymol. 242, 56-65) using a coupling
reaction method. Specifically, the reaction was performed as a two-step
chemical reaction. First, ganglioside existing on the membrane surface of
10 ml of the liposome obtained in Example 2 was added to 43 mg of sodium
metaperiodate dissolved in 1 ml of a TAPS buffer solution (pH8.4),
followed by 2 hours of agitation at room temperature to perform periodate
oxidation. The resultant was subjected to ultrafiltration using an XM300
membrane and a PBS buffer solution (pH8.0) so that 10 ml of the thus
oxidized liposome was obtained. 20 mg of human serum albumin (HSA) was
added to the liposome solution, followed by 2 hours of agitation at
25° C. Next, 100 μl of 2M NaBH3CN was added to PBS (pH8.0)
and then the solution was agitated overnight at 10° C. Thus, HSA
was bound by the coupling reaction between ganglioside on the liposome
and HSA. The resultant was then subjected to ultrafiltration using an
XM300 membrane and a CBS buffer solution (pH8.5), so that 10 ml of an
HSA-bound liposome solution was obtained.

Example 4

Preparation of Sugar Chain

[0348]Sugar chains listed in Table 4 below were used.

[0349]The mass of each sugar chain was measured and then pretreated for
use in the following Example 5. When a combination of two or more sugar
chains was used, these sugar chains were mixed with each other.

[0350]50 μg of each sugar chain prepared in Example 4 was added to 0.5
ml of an aqueous solution in which 0.25 g of NH4HCO3 had been
dissolved, followed by 3 days of agitation at 37° C. The resultant
was filtered with a 0.45-μm filter to complete the amination reaction
of the reducing termini of the sugar chains. Thus, 50 μg of a
glycosylamine compound of each sugar chain was obtained. Next, 1 mg of a
cross-linking reagent 3,3'-dithiobis(sulfo succinimidyl propionate
(DTSSP; Pierce Co., U.S.A.) was added to 1 ml of the liposome solution (a
portion of the liposome solution) obtained in Example 3. The solution was
agitated for 2 hours at 25° C. and then agitated overnight at
7° C. The resultant was subjected to ultrafiltration using an
XM300 membrane and a CBS buffer solution (pH8.5), so that 1 ml of
liposome was obtained on which DTSSP was bound to HSA on the liposome.
Next, 50 μg of the above glycosylamine compound was added to the
liposome solution. The solution was agitated for 2 hours at 25° C.
and then agitated overnight at 7° C. The resultant was subjected
to ultrafiltration using an XM300 membrane and a PBS buffer solution
(pH7.2) so as to bind the glycosylated amine compound to DTSSP on the
liposome membrane-surface-bound human serum albumin. As a result, as
listed in Table 2, liposomes (2 ml each) (total amount of lipid: 2 mg,
total amount of protein: 200 μg, and average particle diameter: 100
nm) were obtained, each of which is prepared by binding of a sugar chain
and human serum albumin, and the liposome.

[0351]Table 10 below shows the results of binding of each sugar chain onto
liposome membrane-surface-bound human serum albumin (HSA). Unless
otherwise clearly specified, binding of these sugar chains onto liposome
membrane-surface-bound human serum albumin was performed with a method
and the conditions similar to those in Example 5.

[0352]To prepare a liposome as a sample for comparison, 1 mg of a
cross-linking reagent 3,3'-dithiobis(sulfosuccinimidyl propionate (DTSSP;
Pierce Co., U.S.A.) was added to 1 ml of the liposome solution (a portion
of the liposome solution) obtained in Example 3. The solution was
agitated for 2 hours at 25° C. and then agitated overnight at
7° C. The resultant was subjected to ultrafiltration using an
XM300 membrane and a CBS buffer solution (pH8.5), so that 1 ml of
liposome in which DTSSP was bound to HSA on the liposome was obtained.
Next, 13 mg of tris(hydroxymethyl)aminomethane (Wako Co., Japan) was
added to the liposome solution. The solution was agitated for 2 hours at
25° C. and then agitated overnight at 7° C. The resultant
was subjected to ultrafiltration using an XM300 membrane and a PBS buffer
solution (pH7.2) so as to bind tris(hydroxymethyl)aminomethane to DTSSP
on the liposome membrane-surface-bound human serum albumin. Because of
the presence of 13 mg of tris(hydroxymethyl)aminomethane, which was
already an extremely excessive amount in this step, hydrophilization of
the liposome membrane-surface-bound human serum albumin (HSA) was also
completed simultaneously. As a result, the final product, 2 ml of
liposome (abbreviated name: TRIS) (total amount of lipid; 2 mg, total
amount of protein: 200 μg, and average particle diameter: 100 nm) as a
sample for comparison was obtained via binding of hydrophilized
tris(hydroxymethyl)aminomethane, human serum albumin, and the liposome.

[0354]Liposomes were prepared using cholic acid dialysis. Specifically,
dipalmitoylphosphatidylcholine, cholesterol, dicetylphosphate,
ganglioside (containing 100% GM1 as a glycolipid sugar chain), and
dipalmitoylphosphatidylethanol amine were mixed at a molar ratio of
35:40:5:15:5 so that the total amount of lipid was 45.6 mg. 46.9 mg of
sodium cholate was added to the mixture and then the resultant was
dissolved in 3 ml of a chloroform/methanol solution. The solution was
evaporated and then the precipitate was dried in vacuum, so that a lipid
membrane was obtained. The thus obtained lipid membrane was suspended in
3 ml of a TAPS buffer solution (pH8.4) and then the suspension was
ultrasonicated. Thus, 3 ml of a transparent micelle suspension was
obtained. A PBS buffer solution (pH7.2) was added to the micelle
suspension to a total amount of 10 ml. The micelle suspension was
subjected to ultrafiltration using a PM10 membrane (Amicon Co., U.S.A.)
and a TAPS buffer solution (pH8.4). Thus, 10 ml of a homogeneous
suspension of unhydrophilized liposome particles was prepared. 5 mg of
Bolton-Hunter Reagent (BHR; Pierce Co., U.S.A.) was added to the liposome
solution, so as to perform 2 hours of reaction at 25° C. and then
to perform 4 hours of reaction at 7° C.
Dipalmitoylphosphatidylethanol amine was thus modified with BH and then
the resultant was subjected to ultrafiltration using a PBS buffer
solution (PH7.2). As a result, 10 ml of liposome (abbreviated name:
EEGM1-BH) (total amount of lipid: 45.6 mg and average particle diameter:
100 nm) was obtained as a sample for comparison.

[0355]Sugar chain-bound liposomes prepared by the means of Example 5 were
separately subjected to hydrophilization of the HSA protein surfaces on
the liposomes as described in the following procedures. 13 mg of
tris(hydroxymethyl)aminomethane was added to 2 ml of each sugar
chain-bound liposome. The solution was agitated for 2 hours at 25°
C. and then agitated overnight at 7° C. The resultant was
subjected to ultrafiltration using an XM300 membrane and a PBS buffer
solution (pH7.2) and then unreacted substances were removed. As a result,
the final product, 2 ml of each hydrophilized sugar chain-bound liposome
complex (total amount of lipid; 2 mg, total amount of protein: 200 μg,
and average particle diameter: 100 nm) was obtained.

[0356]The in vitro binding activity of each sugar chain-bound liposome
complex (prepared by the means of Example 5 and Example 6); that is, the
activity of binding to a cell surface molecule such as a lectin was
determined in an inhibition experiment using a lectin-immobilized
microplate according to a standard method (Yamazaki, N. (1999) Drug
Delivery System, 14, 498-505). Specifically, a lectin (e.g., E-selectin;
R&D Systems Co., U.S.A.; the lectin can be varied according to target
organs) was immobilized on a 96-well microplate. 0.1 μg of
biotinylated fucosylated fetuin (ligand for comparison) and each of
various sugar chain-bound liposome complexes (amounts of protein: 0.01
μg, 0.04 μg, 0.11 μg, 0.33 μg, and 1 μg) varying in
concentration were added to the lectin-immobilized plate, followed by 2
hours of incubation at 4° C. After 3 times of washing with PBS
(pH7.2), horseradish peroxidase (HRPO)-conjugated streptavidin was added
to the resultants. Incubation was further performed at 4° C. for 1
hour and then the resultants were washed 3 times with PBS (pH7.2). A
peroxidase substrate was added, the resultants were allowed to stand at
room temperature, and then absorbance was measured at 405 nm using a
microplate reader (Molecular Devices Corp., U.S.A.). Fucosylated fetuin
was biotinylated as follows. Treatment was performed with a
sulfo-NHS-biotin reagent (Pierce Co., U.S.A.) and then purification was
performed with Centricon-30 (Amicon Co., U.S.A.). HRPO-conjugated
streptavidin was prepared by oxidation of HRPO and conjugation of
streptavidin via reductive amination using NaBH3CN. The
determination results were subjected to the following treatment and
calculation.

[0357]FIGS. 3 to 6 are schematic diagrams as to IC20 to IC50. FIG. 2 shows
Graph 1 showing the results of an experiment conducted for Sample LY-1
and IC50.

[0358]The graph shown in FIG. 2 is a graph of sample LY-1 and IC50 series
produced based on Table 16 and Table 17.

TABLE-US-00026
TABLE 16
Values of sample LY-1 and IC50 at each concentration
Concentration
0.01 0.04 0.11 0.33 1
Average value of sample LY-1 0.144 0.142 0.126 0.110 0.073
Ratio of average value of sample 0.739 0.715 0.562 0.414 0.060
LY-1*
Ratio of y coordinate of IC50 series 0.500 0.500 0.500 0.500 0.500
*"Ratio of average value of sample LY-1" or "Ratio of y coordinate of IC50
series" is the ratio of the same with respect to 1 (a difference between
a "hot" value and a "cold" value of Control is determined to be "1.")

TABLE-US-00027
TABLE 17
Control
Hot Cold
0.171 0.067

[0359]Graph 1 was produced based on Table 1. X axis is expressed using a
logarithmic scale. Each point on the line graph represents the ratio of
the average value (of values measured at each concentration (horizontal
axis)) of sample LY-1. The control value differs depending on samples. To
facilitate comparison, the ratios (with respect to 1 (a difference
between a "hot" value and a "cold" value of Control is determined to be
"1.")) are plotted on the longitudinal axis of the graph. The X
coordinate of the intersection point of the Sample LY-1 graph and the
IC50 series graph is the value of IC50. The intersection point is present
on a line containing coordinate 1 (0.11, 0.562) and coordinate 2 (0.33,
0.414) and is represented by the formula: y=-0.673x+0.636. In the case of
y=0.5 (the formula of IC50 series), X coordinate of the intersection
point of two lines is 0.202. This value is divided by 69000 (the
molecular weight of protein) and then the product is further divided by
300 (the number of protein per liposome). The result is 9.76E-09.

[0364]A chloramine T (Wako Pure Chemical Co., Japan) solution and a sodium
disulfite solution were each prepared at 3 mg/ml and 5 mg/ml when they
were used. The sugar chain-bound liposomes and
tris(hydroxymethyl)aminomethane-bound liposomes prepared in Example 6
were separately added (50 μl each) to Eppen tubes. Subsequently, 15
μl of 125I-NaI (NEN Life Science Product, Inc. U.S.A.) and 10
μl of a chloramine T solution were added to perform reaction. 10 μl
of a chloramine T solution was added every 5 minutes. At 15 minutes after
repeating twice this procedure, 100 μl of sodium disulfite was added
as a reducing agent so as to stop the reaction. Next, the resultants were
placed on a Sephadex G-50 (Pharmacia Biotech. Sweden) column for
chromatography and then subjected to elution with PBS, so that labeled
products were purified. Finally, an unlabeled-liposome complex was added
and then specific activity (4×106 Bq/mg protein) was adjusted.
Thus, 125-labeled liposome solutions were obtained.

Example 9

Measurement of the Amounts of Various Sugar Chain-Bound Liposome Complexes
Transferred from the Intestinal Tract into Blood in Mice

[0365]0.2 ml of each 125I-labeled sugar chain-bound and
tris(hydroxymethyl)aminomethane-bound liposome complex prepared in
Example 6 was forcedly administered intraintestinally using oral sonde
for mice to male ddY mice (7-week-old) that had been fasted overnight
(excluding water), so that the protein amount was 3 μg/mouse. 10
minutes later, 1 ml of blood was collected from inferior vena cava of
each mouse under Nembutal anesthesia. 125I radioactivity in blood
sample was then measured using a gamma counter (Alola ARC300).
Furthermore, to examine the in vivo stability of various liposome
complexes, the serum of each blood was re-chromatographed using Sephadex
G-50. In all cases, most of radioactivity was observed in
high-molecular-weight void fractions. Various liposome complexes also had
stability in vivo. In addition, each amount of radioactivity that had
been transferred from the intestinal tract into blood is represented by
the proportion of radioactivity per ml of blood (% dose/ml blood) with
respect to the total radioactivity administered. The results are shown in
Table 19 below.

[0366]Of the sugar chain-bound liposomes prepared in the above Examples,
liposomes administered herein were: sugar chain-bound liposomes
(hereinafter, sugar chain+liposome) and liposomes to which no sugar chain
had been bound (hereinafter, sugar chain-liposome). The sugar
chain+liposome and the sugar chain-liposome were separately administered
to normal mice via intravenous injection or oral administration for the
purpose of evaluating the accumulation of such liposome in each organ of
the mice.

[0367]The experiment was conducted for normal mice and cancer-bearing
mice. The procedures are as described below. The distribution amounts of
various sugar chain-modified liposomes and liposomes not modified with
sugar chains in each tissue were measured as follows. Mice used for this
experiment were: normal mice; and male ddY mice (7-week-old) to which
Ehrlich ascites tumor (EAT) cells (approximately 2×107) had
been transplanted subcutaneously into the thighs and then the cancerous
tissue had grown to 0.3 g to 0.6 g (grown for 6 to 8 days). 0.2 ml each
of various 125I-labeled liposomes prepared in Example 8 was
administered to these mice via oral administration or injection via tail
vein so that the protein amount was 3 μg/mouse. At 10 or 5 minutes
later, tissues (blood, liver, heart, lungs, pancreas, brain, cancerous
tissue, inflammatory tissue around cancer, small intestine, large
intestine, lymph node, bone marrow, kidney, spleen, thymus gland, and
muscle) were excised. The radioactivity of each tissue was measured using
a gamma counter (Aloka ARC 300). In addition, the amount of radioactivity
distributed in each tissue was obtained by measuring the proportion of
radioactivity per gram of each tissue (% dose/g of each tissue) with
respect to the total radioactivity administered. After oral
administration or intravenous administration of sugar chain-modified
liposomes and liposomes (standard liposome) to which
tris(hydroxymethyl)aminomethane had been bound instead of a sugar chain,
the ratio (magnification) of an average measured value (obtained by
averaging the measured values of the sugar chain-modified liposome
delivered into blood or each tissue of four mice) to an average measured
value (obtained by averaging the values of the standard liposome
delivered into blood or each tissue of four mice) was calculated. Thus,
transferability into blood and tropism (targeting) for each organ after
oral administration were evaluated according to the definition in Table
20A. The results are shown in Table 20B below.

[0368]Table 14 and Table 15B show the evaluation results showing the
effects of accumulating radioactive sugar chain liposomes in each tissue
when various radioactive sugar chain-modified liposomes were administered
to mice via oral administration and intravenous injection. These results
demonstrate that the sugar chain-modified liposomes can achieve highly
efficient accumulation and delivery of drugs, fluorescent substances,
radiolabeled substances, or the like via active targeting to parts
affected by diseases or various organs with the use of the functions of a
sugar chain as a ligand. Therefore, the sugar chain-modified liposomes of
the present invention can visualize accumulation in a target tissue such
as a tumor. Hence, according to the present invention, a delivery vehicle
for use as therapeutic drugs and a delivery vehicle for use as a research
reagent, a diagnostic agent, or the like are also provided.

Example 11

Preparation of Anticancer Agent Doxorubicin-Encapsulated Liposome

[0369]A liposome was prepared by the techniques of the previous report
(Yamazaki, N., Kodama, M. and Gabius, H.-J. (1994) Methods Enzymol. 242,
56-65) using improved cholic acid dialysis. Specifically,
dipalmitoylphosphatidylcholine, cholesterol, dicetylphosphate,
ganglioside, and dipalmitoylphosphatidylethanol amine were mixed at a
molar ratio of 35:40:5:15:5 so that the total amount of lipid was 45.6
mg. 46.9 mg of sodium cholate was added to the mixture and then the
resultant was dissolved in 3 ml of a chloroform/methanol solution. The
solution was evaporated and then the precipitate was dried in vacuum,
thereby obtaining a lipid membrane. The thus obtained lipid membrane was
suspended in 10 ml of a TAPS buffered saline solution (pH8.4) and then
the resultant was ultrasonicated, so that 10 ml of a transparent micelle
suspension was obtained. An anticancer agent doxorubicin that had been
completely dissolved in a TAPS buffer solution (pH8.4) to a concentration
of 3 mg/1 ml was slowly added dropwise to the micelle suspension while
agitating the suspension. After homogeneously mixing the suspension, the
doxorubicin-containing micelle suspension was subjected to
ultrafiltration using a PM10 membrane (Amicon Co., U.S.A.) and a TAPS
buffered saline solution (pH8.4) so that 10 ml of a homogeneous
anticancer agent doxorubicin-encapsulated liposome particle suspension
was prepared.

[0371]10 ml of the anticancer agent doxorubicin-encapsulated liposome
solution prepared in Example 11 was subjected to ultrafiltration using an
XM300 membrane (Amicon Co., U.S.A.) and a CBS buffer solution (pH8.5).
The pH of the solution was adjusted to 8.5. Next, 10 ml of a
cross-linking reagent bis(sulfo succinimidyl)suberate (BS3; Pierce Co.,
U.S.A.) was added. The solution was agitated for 2 hours of agitation at
25° C. and then agitated overnight at 7° C. Thus, chemical
binding reaction between lipid dipalmitoylphosphatidylethanol amine on
the liposome membrane and BS3 was completed. Subsequently, the liposome
solution was subjected to ultrafiltration using an XM300 membrane and a
CBS buffer solution (pH8.5). Next, 40 mg of
tris(hydroxymethyl)aminomethane dissolved in 1 ml of a CBS buffer
solution (pH8.5) was added to 10 ml of the liposome solution. The
solution was agitated for 2 hours at 25° C. and then agitated
overnight at 7° C. Thus, chemical binding reaction between BS3
bound to the lipids on the liposome membrane and
tris(hydroxymethyl)aminomethane was completed. Therefore, the hydroxy
group of tris(hydroxymethyl)aminomethane was coordinated on the lipid
dipalmitoylphosphatidylethanol amine of the anticancer agent
doxorubicin-encapsulated liposome membrane, so that the liposome membrane
surface was hydrated and hydrophilized.

[0372]Human serum albumin (HSA) was bound onto a liposome membrane surface
according to the technique of the previous report (Yamazaki, N., Kodama,
M. and Gabius, H.-J. (1994) Methods Enzymol. 242, 56-65) using a coupling
reaction method. Specifically, the reaction was performed as a two-step
chemical reaction. First, ganglioside existing on the membrane surface of
10 ml of the liposome obtained in Example 2 was added to 43 mg of sodium
metaperiodate dissolved in 1 ml of a TAPS buffer solution (pH8.4),
followed by 2 hours of agitation at room temperature to perform periodate
oxidation. The resultant was subjected to ultrafiltration using an XM300
membrane and a PBS buffer solution (pH8.0) so that 10 ml of the thus
oxidized liposome was obtained. 20 mg of human serum albumin (HSA) was
added to the liposome solution, followed by 2 hours of agitation at
25° C. Next, 100 μl of 2M NaBH3CN was added to PBS (pH8.0)
and then the solution was agitated overnight at 10° C. Thus, HSA
was bound by the coupling reaction between ganglioside on the liposome
and HSA. The resultant was then subjected to ultrafiltration using an
XM300 membrane and a CBS buffer solution (pH8.5), so that 10 ml of an
HSA-bound anticancer agent doxorubicin-encapsulated liposome solution was
obtained.

Example 14

Preparation of Sugar Chain

[0373]Sugar chains were prepared by the procedures similar to those in
Example 4.

[0374]50 μg of each sugar chain prepared in Example 14 was added to 0.5
ml of an aqueous solution in which 0.25 g of NH4HCO3 had been
dissolved, followed by 3 days of agitation at 37° C. The resultant
was filtered with a 0.45-μm filter to complete the amination reaction
of the reducing termini of the sugar chains. Thus, 50 μg of a
glycosylamine compound of each sugar chain was obtained. Next, 1 mg of a
cross-linking reagent 3,3'-dithiobis(sulfo succinimidyl propionate
(DTSSP; Pierce Co., U.S.A.) was added to 1 ml of the anticancer agent
doxorubicin-encapsulated liposome solution (a portion of the liposome
solution) obtained in Example 13. The solution was agitated for 2 hours
at 25° C. and then agitated overnight at 7° C. The
resultant was subjected to ultrafiltration using an XM300 membrane and a
CBS buffer solution (pH8.5), so that 1 ml of liposome was obtained on
which DTSSP had been bound to HSA on the liposome. Next, 50 μg of the
glycosylamine compound of the above sugar chain was added to the liposome
solution. The solution was agitated for 2 hours at 25° C. and then
agitated overnight at 7° C. The resultant was subjected to
ultrafiltration using an XM300 membrane and a PBS buffer solution (pH7.2)
so as to bind each sugar chain to DTSSP on the liposome
membrane-surface-bound human serum albumin. Next, 13 mg of
tris(hydroxymethyl)aminomethane (Wako Co., Japan) was added to the
liposome solution. The solution was agitated for 2 hours at 25° C.
and then agitated overnight at 7° C. The resultant was subjected
to ultrafiltration using an XM300 membrane and a PBS buffer solution
(pH7.2), so as to bind the glycosylated amine compound to DTSSP on the
liposome membrane-surface-bound human serum albumin. As a result, a
liposome was obtained, in which tris(hydroxymethyl)aminomethane, human
serum albumin, and the liposome were bound and the linker protein (HSA)
was hydrophilized. As a result, 2 ml of an anticancer agent
doxorubicin-encapsulated liposome (total amount of lipid: 2 mg and total
amount of protein: 200 μg) was obtained, in which each sugar chain,
human serum albumin, and the liposome were bound and the linker protein
(HSA) was hydrophilized.

[0376]To prepare an anticancer agent doxorubicin-encapsulated liposome as
a sample for comparison, 1 mg of a cross-linking reagent
3,3'-dithiobis(sulfosuccinimidyl propionate (DTSSP; Pierce Co., U.S.A.)
was added to 1 ml of the anticancer agent doxorubicin-encapsulated
liposome solution (a portion of the liposome solution) obtained in
Example 13. The solution was agitated for 2 hours at 25° C. and
then agitated overnight at 7° C. The resultant was subjected to
ultrafiltration using an XM300 membrane and a CBS buffer solution
(pH8.5), so that 1 ml of liposome was obtained in which DTSSP was bound
to HSA on the liposome and the linker protein (HSA) was hydrophilized.
Next, 13 mg of tris(hydroxymethyl)aminomethane (Wako Co., Japan) was
added to the liposome solution. The solution was agitated for 2 hours at
25° C. and then agitated overnight at 7° C. The resultant
was subjected to ultrafiltration using an XM300 membrane and a PBS buffer
solution (pH 7.2) so as to bind the glycosylation amine compound to DTSSP
on the liposome membrane-surface-bound human serum albumin. As a result,
2 ml of an anticancer agent doxorubicin-encapsulated liposome
(abbreviated name: DX-TRIS) (total amount of lipid; 2 mg and total amount
of protein: 200 μg) as a sample for comparison was obtained, in which
tris(hydroxymethyl)aminomethane, human serum albumin, and the liposome
were bound and the linker protein (HSA) was hydrophilized.

[0378]Sugar chain-bound liposomes prepared by the means of Example 15 were
separately subjected to hydrophilization of the HSA protein surfaces on
the liposomes as described in the following procedures. 13 mg of
tris(hydroxymethyl)aminomethane was added to 2 ml of each sugar
chain-bound liposome. The solution was agitated for 2 hours at 25°
C. and then agitated overnight at 7° C. The resultant was
subjected to ultrafiltration using an XM300 membrane and a PBS buffer
solution (pH7.2) and then unreacted substances were removed. As a result,
the final product, 2 ml of each hydrophilized sugar chain-bound liposome
complex (total amount of lipid; 2 mg, total amount of protein: 200 μg,
and average particle diameter: 100 nm) was obtained.

[0379]The in vitro lectin binding activity of each sugar chain-bound
liposome complex (prepared by the means of Example 16) was determined in
an inhibition experiment using a lectin-immobilized microplate according
to a standard method (Yamazaki, N. (1999) Drug Delivery System, 14,
498-505). Specifically, a lectin (e.g., E-selectin; R&D Systems Co.,
U.S.A.) was immobilized on a 96-well microplate. 0.1 μg of
biotinylated fucosylated fetuin (ligand for comparison) and each of
various sugar chain-bound liposome complexes (amounts of protein: 0.01
μg, 0.04 μg, 0.11 μg, 0.33 μg, and 1 μg) varying in
concentration were added to the lectin-immobilized plate, followed by 2
hours of incubation at 4° C. After 3 times of washing with PBS
(pH7.2), horse radish peroxidase (HRPO)-conjugated streptavidin was added
to the resultants. Incubation was further performed at 4° C. for 1
hour and then the resultants were washed 3 times with PBS (pH7.2).
Peroxidase substrate was added, the resultants were allowed to stand at
room temperature, and then absorbance was measured at 405 nm using a
microplate reader (Molecular Devices Corp., U.S.A.). Fucosylated fetuin
was biotinylated as follows. Treatment was performed with a
sulfo-NHS-biotin reagent (Pierce Co., U.S.A.) and then purification was
performed with Centricon-30 (Amicon Co., U.S.A.). HRPO-conjugated
streptavidin was prepared by oxidation of HRPO and conjugation of
streptavidin via reductive amination using NaBH3CN. The measurement
results are as shown in Table 21 below.

[0383]A chloramine T (Wako Pure Chemical Co., Japan) solution and a sodium
disulfite solution were each prepared at 3 mg/ml and 5 mg/ml when they
were used. The sugar chain-bound liposomes and
tris(hydroxymethyl)aminomethane-bound liposomes prepared in Example 16
were separately added (50 μl each) to Eppen tubes. Subsequently, 15
μl of 125I-NaI (NEN Life Science Product, Inc. U.S.A.) and 10
μl of a chloramine T solution were added to perform reaction. 10 μl
of the chloramine T solution was added every 5 minutes. At 15 minutes
after repeating twice this procedure, 100 μl of sodium disulfite was
added as a reducing agent so as to stop the reaction. Next, the
resultants were placed on a Sephadex G-50 (Pharmacia Biotech. Sweden)
column for chromatography and then subjected to elution with PBS, so that
labeled products were purified. Finally, an unlabeled-liposome complex
was added and then specific activity (4×106 Bq/mg protein) was
adjusted. Thus, 125I-labeled liposome solutions were obtained.

Example 19

Measurement of the Amounts of Various Sugar Chain-Bound Liposome Complexes
that Transferred from the Intestinal Tract into Blood in Mice

[0384]0.2 ml of each 125I-labeled sugar chain-bound and
tris(hydroxymethyl)aminomethane-bound liposome complex prepared in
Example 17 was forcedly administered intraintestinally using oral sonde
for mice to male ddY mice (7-week-old) that had been fasted overnight
(excluding water), so that the amount of protein administered herein was
3 μg/mouse. 10 minutes later, 1 ml of blood was collected from
inferior vena cava of each mouse under Nembutal anesthesia. 125I
radioactivity in blood was then measured using a gamma counter (Alola
ARC300). Furthermore, to examine the in vivo stability of various
liposome complexes, the serum of each blood was re-chromatographed using
Sephadex G-50. In all cases, most of radioactivity was observed in
high-molecular-weight void fractions. Various liposome complexes also had
stability in vivo. In addition, each amount of radioactivity that had
been transferred from the intestinal tract into blood is represented by
the proportion of radioactivity per ml of blood (% dose/ml blood) with
respect to the total radioactivity administered. The results are shown in
Table 22.

[0385]Of the sugar chain-bound liposomes prepared in the above Examples,
liposomes administered herein are: sugar chain-bound liposomes
(hereinafter, sugar chain+liposome) and liposomes to which no sugar chain
had been bound (hereinafter, sugar chain-liposome). The sugar
chain+liposome and the sugar chain-liposome were separately administered
to normal mice via oral administration for the purpose of evaluating the
accumulation of such liposome in each organ of the mice.

[0386]Liposome solutions that had been prepared in advance were
administered to mice via oral administration and then all the organs were
each excised. Each organ was prepared as a tissue homogenate using a 1%
Triton X solution and a HG30 homogenizer (Hitachi Koki Co., Ltd.).
Liposomes contained in the tissue homogenates were extracted using 100%
methanol and chloroform. Regarding the amount of a liposome, the
fluorescence intensity of FITC bound to the liposome was measured using a
fluorescent microplate reader Biolumin960 (Molecular Dynamics) at 490 nm
of excitation and 520 nm of emission. Regarding data obtained via oral
administration, the results obtained via oral administration alone are
shown. Regarding the other organs (not obtained via oral administration),
the results obtained via intravenous injection are shown. In the case of
oral administration, vehicles that had transferred into blood as a result
of oral administration showed similar tendency to that in the case of
intravenous injection.

[0387]A liposome was prepared using cholic acid dialysis. Specifically,
dipalmitoylphosphatidylcholine, cholesterol, dicetylphosphate,
ganglioside, and dipalmitoylphosphatidylethanol amine were mixed at a
molar ratio of 35:40:5:15:5 so that the total amount of lipid was 45.6
mg. 46.9 mg of sodium cholate was added to the mixture and then the
resultant was dissolved in 3 ml of a chloroform/methanol solution. The
solution was evaporated and then the precipitate was dried in vacuum,
thereby obtaining a lipid membrane. The thus obtained lipid membrane was
suspended in 10 ml of TAPS buffered saline solution (pH8.4) and then the
resultant was ultrasonicated, so that 10 ml of a transparent micelle
suspension was obtained. An anticancer agent doxorubicin that had been
completely dissolved in a TAPS buffer solution (pH8.4) to a concentration
of 3 mg/1 ml was slowly added dropwise to the micelle suspension while
agitating the suspension. After homogeneously mixing the suspension, the
doxorubicin-containing micelle suspension was subjected to
ultrafiltration using a PM10 membrane (Amicon Co., U.S.A.) and a TAPS
buffered saline solution (pH8.4) so that 10 ml of a homogeneous
anticancer agent doxorubicin-encapsulated liposome particle suspension
was prepared. The particle diameter and zeta potential of the anticancer
agent doxorubicin-encapsulated liposome particles in the thus obtained
physiological saline suspension (37° C.) were measured using a
zeta potential-particle diameter-molecular weight measurement apparatus
(Model Nano ZS, Malvern Instruments Ltd., UK). As a result, the particle
diameter ranged from 50 nm to 350 nm and the zeta potential ranged from
-30 mV to -10 mV.

[0389]Cancer-bearing mice were produced as follows. The hair on the back
of each ddY 7-week-old mouse (male and body weight: 35 g to 40 g) was
shaved using an electric shaver and then Ehrlich Ascites Tumor
(approximately 5×106 cells/mouse) was subcutaneously
transplanted. The mice were grown and observed for 10 days. The mice in
which cancer cells had successfully survived and grown were selected and
then used for the experiment. Drug administration and measurement of the
volume of cancer were performed as follows. Two types of group were
prepared: a group to which doxorubicin-encapsulated liposome No. 155 had
been administered, in which the concentration of doxorubicin
(encapsulated as a drug to be administered) had been adjusted at 0.0625
mg/kg; and a group to which a physiological saline solution had been
administered as a control. The liposome was administered through
injection via tail vein to cancer-bearing mice 4 times a week for 2
weeks. The major diameter of cancer and the minor diameter of cancer were
measured using a micrometer caliper. The measurement was initiated on day
10 after transplantation of cancer cells and performed twice a week for 4
weeks. The volume of cancer that had grown was calculated by the
following formula.

Volume of cancer (mm3)=(major diameter+minor diameter2)/2

[0390]FIG. 1 shows the results. Changes in the volume of tumor that had
grown were compared in each of the group to which
doxorubicin-encapsulated liposome No. 155 had been administered and the
group to which the physiological saline solution had been administered as
a control. Although the extremely small dose of the liposome, the effect
of suppressing cancer growth was exerted significantly in the group to
which doxorubicin-encapsulated liposome No. 155 had been administered,
such that cancer growth was suppressed from the initiation of
administration. The two groups were compared in terms of cancerous tissue
size (volume) on day 34 after transplantation; that is the final
measurement day. A more significant antiproliferative effect was observed
in the group to which doxorubicin-encapsulated liposome No. 155 had been
administered compared with that in the group to which the physiological
saline solution had been administered. These results demonstrate that the
antineoplastic agent-encapsulated and sugar chain-modified liposome has a
strong anticancer effect even when an extremely small dose of the agent
is administered (FIG. 7).

(2) Fluorescent Microscopic Observation of the Transfer of Doxorubicin to
Cancerous Tissue when Doxorubicin was Administered Through Injection Via
Tail Vein

[0392]Fluorescent microscopic observation was performed as follows. The
skin of a cancer site of each cancer-bearing mouse was excised to expose
the cancerous tissue. The cancer site was fixed on a slide glass. Each
mouse was placed on the stage of a fluorescence microscope. Blood vessels
around the cancerous tissue were searched, so that a position at which
the blood vessel image could be clearly observed was determined. 0.2 ml
of doxorubicin-encapsulated liposome No. 155 (lipid concentration: 2
mg/mL and doxorubicin concentration: 0.025 mg/mL) was administered
through injection via tail vein. Immediately after administration,
observation of the accumulation of doxorubicin into the cancerous tissue
was initiated under a fluorescence microscope. An inhibition experiment
involving pre-administration of a modified sugar was conducted as
follows. 0.2 mL of a modified sugar chain (α1-6mannobiose) solution
(60 mM) was administered at 5 minutes before administration of
doxorubicin-encapsulated liposome No. 155. Observation was performed by
the method similar to the above. Photograph 1 shows the result.
Immediately after administration of doxorubicin-encapsulated liposome No.
155, doxorubicin fluorescence was observed in the blood vessels in the
vicinity of the cancerous tissue. At 5 minutes after administration, red
doxorubicin fluorescence was observed in the blood vessel wall part.
Thereafter, the transfer of doxorubicin to the relevant tissues with the
course of time was observed. Two hours later, doxorubicin fluorescence
was observed within the tumor tissues in the periphery of cancerous blood
vessels. Pre-administration of the modified sugar chain resulted in
complete block of the accumulation of doxorubicin-encapsulated liposome
No. 155 into the tumor tissues. Hence, fluorescence was not observed in
the blood vessel walls from the time point immediately after
administration of doxorubicin-encapsulated liposome No. 155. These
results demonstrate that the sugar chain-modified liposomes can achieve
highly efficient accumulation and delivery of drugs, fluorescent
substances, radiolabeled substances, or the like via active targeting to
parts affected by diseases or various organs with the use of the
functions of a sugar chain as a ligand (FIG. 8).

[0393]FIG. 8 shows fluorescent microscopic photographs showing the effect
of accumulating doxorubicin from tumor blood vessels to tumor tissues and
cells in cancer-bearing mice when doxorubicin-encapsulated liposome No.
155 was administered through injection via tail vein. Green fluorescent
microscopic photographs on the left are of the same tissue or cells. Red
fluorescent microscopic photographs on the right are of the same tumor
tissue or cells. Green (images on the left) shows natural fluorescence of
the blood vessels, tissue, and cells. Red (images on the right) shows
doxorubicin fluorescence (doxorubicin is a fluorescent substance) in the
tumor tissue and the cancer cells. These results demonstrate that the
sugar chain-modified liposomes can achieve highly efficient accumulation
and delivery of drugs, fluorescent substances, radiolabeled substances,
or the like via active targeting to parts affected by diseases or various
organs with the use of the functions of a sugar chain as a ligand.
Therefore, the sugar chain-modified liposome of the present invention can
visualize the accumulation in target tissues such as tumors. Thus,
according to the present invention, in addition to a delivery vehicle for
delivering a drug for treatment, a delivery vehicle to be used as a
reagent for research or a diagnostic agent is provided.

Example 22B

Measurement of Anticancer Effect of Various Sugar Chain-Bound Liposome
Complexes in Cancer-Bearing Mice when the Complexes were Administered Via
Oral Administration

[0395]Cancer-bearing mice were produced as follows. The hair on the back
of each ddY 7-week-old mouse (male and body weight: 35 g to 40 g) was
shaved using an electric shaver and then Ehrlich Ascites Tumor
(approximately 5×106 cells/mouse) was subcutaneously
transplanted. The mice were grown and observed for about 10 days. The
mice in which cancer cells had successfully survived and grown were
selected and then used for the experiment. Drug administration and
measurement of the volume of cancer were performed as follows. Two types
of group were prepared: a group to which doxorubicin-encapsulated
liposome No. 237 had been administered, in which the concentration of
doxorubicin (encapsulated as a drug to be administered) had been adjusted
at 0.375 mg/kg; and a group to which a physiological saline solution had
been administered as a control. The liposome was administered via oral
administration to cancer-bearing mice 4 times a week for 2 weeks. The
major diameter of cancer and the minor diameter of cancer were measured
using a micrometer caliper. The measurement was initiated on day 10 after
transplantation of cancer cells and performed twice a week for 4 weeks.
The volume of cancer that had grown was calculated by the following
formula:

Volume of cancer (mm3)=(major diameter+minor diameter2)/2.

[0396]FIG. 1 shows the results. Changes in the volume of tumor that had
grown were compared in each of the group to which
doxorubicin-encapsulated liposome No. 237 had been administered and the
group to which the physiological saline solution had been administered as
a control. In spite of the extremely small dose, the effect of
suppressing cancer growth was exerted significantly in the group to which
doxorubicin-encapsulated liposome No. 237 had been administered, such
that cancer growth was suppressed from the initiation of administration.
The two groups were compared in terms of cancerous tissue size (volume)
on day 34 after transplantation; that is the final measurement day. A
more significant antiproliferative effect was observed in the group to
which doxorubicin-encapsulated liposome No. 237 had been administered
compared with that in the group to which the physiological saline
solution had been administered. These results demonstrate that the
antineoplastic agent-encapsulated and sugar chain-modified liposome has a
strong anticancer effect even when an extremely small dose is
administered (FIG. 9).

(2) Fluorescent Microscopic Observation of the Transfer of Doxorubicin to
Cancerous Tissue when the Liposome was Administered Via Oral
Administration

[0398]Fluorescent microscopic observation was performed as follows. The
skin of a cancer site of each cancer-bearing mouse was excised to expose
the cancerous tissue. The cancer site was fixed on a slide glass. The
mouse was placed on the stage of a fluorescence microscope. Blood vessels
in the vicinity of the cancerous tissue were searched, so that a position
at which a blood vessel image could be clearly observed was determined.
0.3 ml of doxorubicin-encapsulated liposome No. 237 (lipid concentration:
4 mg/mL and doxorubicin concentration: 0.050 mg/mL) was administered via
oral administration. Immediately after administration, observation of the
accumulation of doxorubicin into the cancerous tissues was initiated
under a fluorescence microscope. An inhibition experiment involving
pre-administration of a modified sugar was conducted as follows. 0.3 mL
of a modified sugar chain (α1-3mannobiose) solution (60 mM) was
administered at 5 minutes before administration of
doxorubicin-encapsulated liposome No. 237. Observation was performed by
the method similar to the above. Photograph 1 shows the result. After
administration of doxorubicin-encapsulated liposome No. 237, the transfer
of doxorubicin to the tissues was observed with the course of time. 6
hours later, doxorubicin fluorescence was observed within the tumor
tissues in the vicinity of cancerous blood vessels. Pre-administration of
the modified sugar chain resulted in the complete block of the
accumulation of doxorubicin-encapsulated liposome No. 237 into the tumor
tissues. Hence, fluorescence was not observed in the blood vessel walls
from the time point immediately after administration of
doxorubicin-encapsulated liposome No. 237. These results demonstrate that
the sugar chain-modified liposomes can achieve highly efficient
accumulation and delivery of drugs, fluorescent substances, radiolabeled
substances, or the like via active targeting to parts affected by
diseases or various organs with the use of the functions of a sugar chain
as a ligand (FIG. 10).

[0399]FIG. 10 shows fluorescent microscopic photographs showing the effect
of accumulating doxorubicin from tumor blood vessels to tumor tissues and
cells in cancer-bearing mice when doxorubicin-encapsulated liposome No.
237 was administered through injection via tail vein. Green fluorescent
microscopic photographs on the left are of the same tumor tissue and
cells. Red fluorescent microscopic photographs on the right are of the
same tumor tissue and cells. Green (images on the left) shows natural
fluorescence of the blood vessel, tissue, and cells. Red (images on the
right) shows doxorubicin fluorescence (doxorubicin is a fluorescent
substance) in the tumor tissue and the cancer cells. These results
demonstrate that the sugar chain-modified liposomes can achieve highly
efficient accumulation and delivery of drugs, fluorescent substances,
radiolabeled substances, or the like via active targeting to parts
affected by diseases or various organs with the use of the functions of a
sugar chain as a ligand. Therefore, the sugar chain-modified liposomes of
the present invention can visualize the accumulation in target tissues
such as tumors.

[0400]Thus, according to the present invention, in addition to a delivery
vehicle for delivering a drug for treatment, a delivery vehicle to be
used as a reagent for research or a diagnostic agent is provided.

Example 23

Preparation of Sugar Chain-Modified Liposome Appropriate for
Administration Based on Rolling Model

[0401]Measurement results (obtained with the use of E-selectin) determined
based on Example 7 was simplified and analyzed as follows using the
rolling model of the present invention.

[0402]Graphs used for calculation of typical IC10, IC20, IC30, IC40, IC50,
and IC60 are shown (typically, see FIGS. 2 to 6).

[0403]Next, the thus obtained IC10 or the like was compared with in vivo
affinity. The results are shown in the following Table. In the Table,
typical results obtained for tumors and inflammation sites are listed.

[0404]As a result, for example, typically in the case of E-selectin, it
was revealed that a threshold value of strong binding and weak binding is
present between approximately IC30 and IC31. The presence of similar
threshold values is expected in the case of other lectins.

[0405]Moreover, it was also revealed that concerning in vitro affinity for
a lectin associated with a desired site, an inhibitory concentration at a
strong binding IC that is approximately IC30 or less is 10-9M or
less (preferably, 5×10-10 or less) and an inhibitory
concentration at a weak binding IC that is approximately IC31 or more is
10-9M or more (preferably, 5×10-8M or more, 10-8M or
more).

[0406]E-selectin is significantly expressed in, when it is administered
via oral administration, the liver, small intestine, large intestine,
lymph node, liver, heart, pancreas, inflammation sites, and cancer sites.
The expression of E-selectin is particularly significant and
characteristic in inflammation sites and cancer sites. It was thus
revealed that E-selectin can be used in in vitro convenient rolling model
assay performed for these organs. Based on these results, delivery
vehicles were prepared as follows.

(1 Preparation of Liposome)

[0407]A liposome was prepared by the techniques of the previous report
(Yamazaki, N., Kodama, M. and Gabius, H.-J. (1994) Methods Enzymol. 242,
56-65) using improved cholic acid dialysis. Specifically,
dipalmitoylphosphatidylcholine, cholesterol, dicetylphosphate,
ganglioside, and dipalmitoylphosphatidylethanol amine were mixed at a
molar ratio of 35:40:5:15:5 so that the total amount of lipid was 45.6
mg. 46.9 mg of sodium cholate was added to the mixture and then the
resultant was dissolved in 3 ml of a chloroform/methanol solution. The
solution was evaporated and then the precipitate was dried in vacuum,
thereby obtaining a lipid membrane. The thus obtained lipid membrane was
suspended in 3 ml of a TAPS buffer solution (pH8.4) and then the
resultant was ultrasonicated, so that a transparent micelle suspension
was obtained. Furthermore, the micelle suspension was subjected to
ultrafiltration using a PM10 membrane (Amicon Co., U.S.A.) and a PBS
buffer solution (pH7.2). Thus 10 ml of homogeneous liposome (average
particle diameter: 100 nm) was prepared.

(2. Hydrophilization of Liposome Lipid Membrane Surface)

[0408]10 ml of the liposome solution prepared in 1 above was subjected to
ultrafiltration using an XM300 membrane (Amicon Co., U.S.A.) and a CBS
buffer solution (pH8.5) and the pH of the solution was adjusted to pH8.5.
Next, 10 ml of a cross-linking reagent bis(sulfosuccinimidyl)suberate
(BS3; Pierce Co., U.S.A.) was added, followed by 2 hours of agitation at
25° C. Subsequently, the solution was further agitated overnight
at 7° C. so as to complete the chemical binding reaction between
lipid dipalmitoylphosphatidylethanol amine on the liposome membrane and
BS3. The liposome solution was then subjected to ultrafiltration using an
XM300 membrane and a CBS buffer solution (pH8.5). Next, 40 mg of
tris(hydroxymethyl)aminomethane dissolved in 1 ml of a CBS buffer
solution (pH8.5) was added to 10 ml of the liposome solution, followed by
2 hours of agitation at 25° C. The solution was then agitated
overnight at 7° C., so as to complete the chemical binding
reaction between BS3 bound to the lipids on the liposome membrane and
tris(hydroxymethyl)aminomethane. Thus, the hydroxy group of
tris(hydroxymethyl)aminomethane was coordinated on the lipid
dipalmitoylphosphatidylethanol amine of the liposome membrane, so that
the liposome membrane surface was hydrated and hydrophilized.

[0409]Human serum albumin (HSA) was bound onto a liposome membrane surface
according to the technique of the previous report (Yamazaki, N., Kodama,
M. and Gabius, H.-J. (1994) Methods Enzymol. 242, 56-65) using a coupling
reaction method. Specifically, the reaction was performed as a two-step
chemical reaction. First, ganglioside existing on the liposome membrane
surface of 10 ml of the liposome obtained in 2 above was added to 43 mg
of sodium metaperiodate dissolved in 1 ml of a TAPS buffer solution
(pH8.4), followed by 2 hours of agitation at room temperature to perform
periodate oxidation. The resultant was subjected to ultrafiltration using
an XM300 membrane and a PBS buffer solution (pH8.0) so that 10 ml of the
thus oxidized liposome was obtained. 20 mg of human serum albumin (HSA)
was added to the liposome solution, followed by 2 hours of agitation at
25° C. Next, 100 μl of 2M NaBH3CN was added to PBS (pH8.0)
and then the solution was agitated overnight at 10° C. Thus, HSA
was bound by the coupling reaction between ganglioside on the liposome
and HSA. The resultant was then subjected to ultrafiltration using an
XM300 membrane and a CBS buffer solution (pH8.5), so that 10 ml of an
HSA-bound liposome solution was obtained.

(4. Preparation of Sugar Chain)

[0410]Sugar chains were prepared by the procedures same as those in
Example 4.

[0411]50 μg of each sugar chain prepared in 4 above was added to 0.5 ml
of an aqueous solution in which 0.25 g of NH4HCO3 had been
dissolved, followed by 3 days of agitation at 37° C. The resultant
was filtered with a 0.45-μm filter to complete the amination reaction
of the reducing termini of the sugar chains. Thus, 50 μg of a
glycosylamine compound of each sugar chain was obtained. Next, 1 mg of a
cross-linking reagent 3,3'-dithiobis(sulfo succinimidyl propionate
(DTSSP; Pierce Co., U.S.A.) was added to 1 ml of the liposome solution (a
portion of the liposome solution) obtained in Example 3. The solution was
agitated for 2 hours at 25° C. and then agitated overnight at
7° C. The resultant was subjected to ultrafiltration using an
XM300 membrane and a CBS buffer solution (pH8.5), so that 1 ml of
liposome was obtained on which DTSSP was bound to HSA on the liposome.
Next 50 μg of the above glycosylamine compound was added to the
liposome solution. The solution was agitated for 2 hours at 25° C.
and then agitated overnight at 7° C. The resultant was subjected
to ultrafiltration using an XM300 membrane and a PBS buffer solution
(pH7.2) so as to bind the glycosylated amine compound to DTSSP on the
liposome membrane-surface-bound human serum albumin. As a result, as
listed in Table 2, liposomes (2 ml each) (total amount of lipid: 2 mg,
total amount of protein: 200 μg, and average particle diameter: 100
nm) were obtained, each of which was prepared by binding of a sugar
chain, human serum albumin, and the liposome. Unless otherwise clearly
specified, binding of these sugar chains onto liposome
membrane-surface-bound human serum albumin were performed by the method
and the conditions similar to those in Example 5.

[0412]To prepare a liposome as a sample for comparison, 1 mg of a
cross-linking reagent 3,3'-dithiobis(sulfosuccinimidyl propionate (DTSSP;
Pierce Co., U.S.A.) was added to 1 ml of the liposome solution (a portion
of the liposome solution) obtained in Example 3. The solution was
agitated for 2 hours at 25° C. and then agitated overnight at
7° C. The resultant was subjected to ultrafiltration using an
XM300 membrane and a CBS buffer solution (pH8.5), so that 1 ml of
liposome in which DTSSP was bound to HSA on the liposome was obtained.
Next, 13 mg of tris(hydroxymethyl)aminomethane (Wako Co., Japan) was
added to the liposome solution. The solution was agitated for 2 hours at
25° C. and then agitated overnight at 7° C. The resultant
was subjected to ultrafiltration using an XM300 membrane and a PBS buffer
solution (pH7.2) so as to bind the glycosylated amine compound to DTSSP
on the liposome membrane-surface-bound human serum albumin. Because of
the presence of 13 mg of tris(hydroxymethyl)aminomethane, which was
already an extremely excessive amount in this step, hydrophilization on
the liposome membrane-surface-bound human serum albumin (HSA) was also
completed simultaneously. As a result, the final product, 2 ml of
liposome (abbreviated name: TRIS) (total amount of lipid; 2 mg, total
amount of protein: 200 μg, and average particle diameter: 100 nm) as a
sample for comparison was obtained via binding of hydrophilized
tris(hydroxymethyl)aminomethane, human serum albumin, and the liposome.

[0413]Sugar chain-bound liposomes prepared by the means of 5.1 above were
separately subjected to hydrophilization of the HSA protein surfaces on
the liposomes as described in the following procedures. 13 mg of
tris(hydroxymethyl)aminomethane was added to 2 ml of each sugar
chain-bound liposome. The solution was agitated for 2 hours at 25°
C. and then agitated overnight at 7° C. The resultant was
subjected to ultrafiltration using an XM300 membrane and a PBS buffer
solution (pH7.2) and then unreacted substances were removed. As a result,
the final product, 2 ml of each hydrophilized sugar chain-bound liposome
complex (total amount of lipid; 2 mg, total amount of protein: 200 μg,
and average particle diameter: 100 nm) was obtained.

(7.)

[0414]As described above, compositions appropriate for delivery to organs
corresponding to E-selectin could be prepared. When these compositions
are actually tested as in the above Examples, it can be confirmed that
they are successfully delivered in vivo to desired organs.

[0415]Since E-selectin is an indicator of an anticancer agent for oral
administration, anticancer agent doxorubicin-encapsulated liposomes were
prepared as follows using optimum compounds based on the rolling model
and then whether or not the compounds actually had antitumor action was
confirmed.

(1. Preparation of Anticancer Agent Doxorubicin-Encapsulated Liposome

[0416]A liposome was prepared by the techniques of the previous report
(Yamazaki, N., Kodama, M. and Gabius, H.-J. (1994) Methods Enzymol. 242,
56-65) using improved cholic acid dialysis. Specifically,
dipalmitoylphosphatidylcholine, cholesterol, dicetylphosphate,
ganglioside, and dipalmitoylphosphatidylethanol amine were mixed at a
molar ratio of 35:40:5:15:5 so that the total amount of lipid was 45.6
mg. 46.9 mg of sodium cholate was added to the mixture and then the
resultant was dissolved in 3 ml of a chloroform/methanol solution. The
solution was evaporated and then the precipitate was dried in vacuum,
thereby obtaining a lipid membrane. The thus obtained lipid membrane was
suspended in 10 ml of a TAPS buffered saline solution (pH8.4) and then
the resultant was ultrasonicated, so that 10 ml of a transparent micelle
suspension was obtained. An anticancer agent doxorubicin that had been
completely dissolved in a TAPS buffer solution (pH8.4) to a concentration
of 3 mg/l ml was slowly added dropwise to the micelle suspension while
agitating the suspension. After homogeneously mixing the suspension, the
doxorubicin-containing micelle suspension was subjected to
ultrafiltration using a PM10 membrane (Amicon Co., U.S.A.) and a TAPS
buffered saline solution (pH8.4) so that 10 ml of a homogeneous
anticancer agent doxorubicin-encapsulated liposome particle suspension
was prepared.

[0418]10 ml of the anticancer agent doxorubicin-encapsulated liposome
solution prepared in 1 above was subjected to ultrafiltration using an
XM300 membrane (Amicon Co., U.S.A.) and a CBS buffer solution (pH8.5).
The pH of the solution was adjusted to 8.5. Next, 10 ml of a
cross-linking reagent bis(sulfo succinimidyl)suberate (BS3; Pierce Co.,
U.S.A.) was added. The solution was agitated for 2 hours at 25° C.
and then agitated overnight at 7° C. Thus, chemical binding
reaction between lipid dipalmitoylphosphatidylethanol amine on the
liposome membrane and BS3 was completed. Subsequently, the liposome
solution was subjected to ultrafiltration using an XM300 membrane and a
CBS buffer solution (pH8.5). Next, 40 mg of
tris(hydroxymethyl)aminomethane dissolved in 1 ml of a CBS buffer
solution (pH8.5) was added to 10 ml of the liposome solution. The
solution was agitated for 2 hours at 25° C. and then further
agitated overnight at 7° C. Thus, chemical binding reaction
between BS3 bound to the lipids on the liposome membrane and
tris(hydroxymethyl)aminomethane was completed. Therefore, the hydroxy
group of tris(hydroxymethyl)aminomethane was coordinated on the lipid
dipalmitoylphosphatidylethanol amine of anticancer agent
doxorubicin-encapsulated liposome membrane, so that the liposome membrane
surface was hydrated and hydrophilized.

[0419]Human serum albumin (HSA) was bound onto a liposome membrane surface
according to the technique of the previous report (Yamazaki, N., Kodama,
M. and Gabius, H.-J. (1994) Methods Enzymol. 242, 56-65) using a coupling
reaction method. Specifically, the reaction was performed as a two-step
chemical reaction. First, ganglioside existing on the membrane surface of
10 ml of the liposomes obtained in 2 above was added to 43 mg of sodium
metaperiodate dissolved in 1 ml of a TAPS buffer solution (pH 8.4),
followed by 2 hours of agitation at room temperature to perform periodate
oxidation. The resultant was subjected to ultrafiltration using an XM300
membrane and a PBS buffer solution (pH 8.0) so that 10 ml of the thus
oxidized liposome was obtained. 20 mg of human serum albumin (HSA) was
added to the liposome solution, followed by 2 hours of agitation at
25° C. Next, 100 μl of 2M NaBH3CN was added to PBS (pH
8.0) and then the solution was agitated overnight at 10° C. Thus,
HSA was bound by the coupling reaction between ganglioside on the
liposome and HSA. The resultant was then subjected to ultrafiltration
using an XM300 membrane and a CBS buffer solution (pH 8.5), so that 10 ml
of an HSA-bound anticancer agent doxorubicin-encapsulated liposome
solution was obtained.

(4. Preparation of Sugar Chain)

[0420]Sugar chains were prepared by procedures similar to those in Example
4.

[0421]50 μg of each sugar chain prepared in 4 above was added to 0.5 ml
of an aqueous solution in which 0.25 g of NH4HCO3 had been
dissolved, followed by 3 days of agitation at 37° C. The resultant
was filtered with a 0.45-μm filter to complete the amination reaction
of the reducing termini of the sugar chains. Thus, 50 μg of a
glycosylamine compound of each sugar chain was obtained. Next, 1 mg of a
cross-linking reagent 3,3'-dithiobis(sulfo succinimidyl propionate
(DTSSP; Pierce Co., U.S.A.) was added to 1 ml of the anticancer agent
doxorubicin-encapsulated liposome solution (a portion of the liposome
solution) obtained in Example 13. The solution was agitated for 2 hours
at 25° C. and then agitated overnight at 7° C. The
resultant was subjected to ultrafiltration using an XM300 membrane and a
CBS buffer solution (pH8.5), so that 1 ml of liposome was obtained on
which DTSSP was bound to HSA on the liposome. Next, 50 μg of the
glycosylamine compound of the above sugar chain was added to the liposome
solution. The solution was agitated for 2 hours at 25° C. and then
agitated overnight at 7° C. The resultant was subjected to
ultrafiltration using an XM300 membrane and a PBS buffer solution (pH7.2)
so as to bind each sugar chain to DTSSP on the liposome
membrane-surface-bound human serum albumin. Next, 13 mg of
tris(hydroxymethyl)aminomethane (Wako Co., Japan) was added to the
liposome solution. The solution was agitated for 2 hours at 25° C.
and then agitated overnight at 7° C. The resultant was subjected
to ultrafiltration using an XM300 membrane and a PBS buffer solution
(pH7.2), so as to bind the glycosylated amine compound to DTSSP on the
liposome membrane-surface-bound human serum albumin. As a result, a
liposome was obtained, in which tris(hydroxymethyl)aminomethane, human
serum albumin, and the liposome were bound and the linker protein (HSA)
was hydrophilized. As a result, 2 ml of an anticancer agent
doxorubicin-encapsulated liposome (total amount of lipid: 2 mg and total
amount of protein: 200 μg) was obtained, in which each sugar chain,
human serum albumin, and the liposome were bound and the linker protein
(HSA) was hydrophilized.

[0423]To prepare a anticancer agent doxorubicin-encapsulated liposome as a
sample for comparison, 1 mg of a cross-linking reagent
3,3'-dithiobis(sulfosuccinimidyl propionate (DTSSP; Pierce Co., U.S.A.)
was added to 1 ml of the anticancer agent doxorubicin-encapsulated
liposome solution (a portion of the liposome solution) obtained in
Example 13. The solution was agitated for 2 hours at 25° C. and
then agitated overnight at 7° C. The resultant was subjected to
ultrafiltration using an XM300 membrane and a CBS buffer solution
(pH8.5), so that 1 ml of liposome in which DTSSP was bound to HSA on the
liposome and the linker protein (HSA) was hydrophilized was obtained.
Next, 13 mg of tris(hydroxymethyl)aminomethane (Wako Co., Japan) was
added to the liposome solution. The solution was agitated for 2 hours at
25° C. and then agitated overnight at 7° C. The resultant
was subjected to ultrafiltration using an XM300 membrane and a PBS buffer
solution (pH7.2) so as to bind the glycosylated amine compound to DTSSP
on the liposome membrane-surface-bound human serum albumin.

[0424]As a result, 2 ml of an anticancer agent doxorubicin-encapsulated
liposome (abbreviated name: DX-TRIS) (total amount of lipid; 2 mg and
total amount of protein: 200 μg) as a sample for comparison was
obtained, in which tris(hydroxymethyl)aminomethane, human serum albumin,
and the liposome were bound and the linker protein (HSA) was
hydrophilized.

[0426]HSA protein surfaces on the sugar chain-bound liposomes prepared by
the means in 5.1 above were hydrophilized by the following procedures. 13
mg of tris(hydroxymethyl)aminomethane was added to 2 ml of each sugar
chain-bound liposome. The resultant was agitated for 2 hours at
25° C. and then agitated overnight at 7° C. Ultrafiltration
was performed using an XM300 membrane and a PBS buffer solution (pH7.2),
so as to remove unreacted products. As a result, the final product, 2 ml
of a hydrophilized sugar chain-bound liposome complex (total amount of
lipid; 2 mg, total amount of protein: 200 μg, and average particle
diameter: 100 nm) was obtained.

[0427]The in vitro lectin binding activity of each sugar chain-bound
liposome complex (prepared by the means of 5.1 and 5.2 above) was
determined in an inhibition experiment using a lectin-immobilized
microplate according to a standard method (Yamazaki, N. (1999) Drug
Delivery System, 14, 498-505). Specifically, a lectin (e.g., E-selectin;
R&D Systems Co., U.S.A.) was immobilized on a 96-well microplate. 0.1
μg of biotinylated fucosylated fetuin (ligand for comparison) and each
of various sugar chain-bound liposome complexes (amounts of protein: 0.01
μg, 0.04 μg, 0.11 μg, 0.33 μg, and 1 μg) varying in
concentration were added to the lectin-immobilized plate, followed by 2
hours of incubation at 4° C. After 3 times of washing with PBS
(pH7.2), horse radish peroxidase (HRPO)-conjugated streptavidin was added
to the resultants. Incubation was further performed at 4° C. for 1
hour and then the resultants were washed 3 times with PBS (pH7.2).
Peroxidase substrate was added, the resultants were allowed to stand at
room temperature, and then absorbance was measured at 405 nm using a
microplate reader (Molecular Devices Corp., U.S.A.). Fucosylated fetuin
was biotinylated as follows. Treatment was performed with a
sulfo-NHS-biotin reagent (Pierce Co., U.S.A.) and then purification was
performed with Centricon-30 (Amicon Co., U.S.A.). HRPO-conjugated
streptavidin was prepared by oxidation of HRPO and conjugation of
streptavidin via reductive amination using NaBH3CN.

[0428]A chloramine T (Wako Pure Chemical Co., Japan) solution and a sodium
disulfite solution were each prepared at 3 mg/ml and 5 mg/ml when they
were used. The sugar chain-bound liposomes and
tris(hydroxymethyl)aminomethane-bound liposomes prepared in 6 were
separately added (50 μl each) to Eppen tubes. Subsequently, 15 μl
of 125I-NaI (NEN Life Science Product, Inc. U.S.A.) and 10 μl of
a chloramine T solution were added to perform reaction. 10 μl of a
chloramine T solution was added every 5 minutes. At 15 minutes after
repeating twice this procedure, 100 μl of sodium disulfite was added
as a reducing agent so as to stop the reaction. Next, the resultants were
placed on a Sephadex G-50 (Pharmacia Biotech. Sweden) column for
chromatography and then subjected to elution with PBS, so that labeled
products were purified. Finally, an unlabeled-liposome complex was added
and then specific activity (4×106 Bq/mg protein) was adjusted.
Thus, 125I-labeled liposome solutions were obtained.

(9. Measurement of the Amounts of Various Sugar Chain-Bound Liposome
Complexes Transferred from the Intestinal Tract into Blood in Mice

[0429]0.2 ml each of 125I-labeled sugar chain-bound and
tris(hydroxymethyl)aminomethane-bound liposome complexes prepared in
Example 17 was forcedly administered intraintestinally using oral sonde
for mice to male ddY mice (7-week-old) that had been fasted overnight
(excluding water), so that the protein amount was 3 μg/mouse. 10
minutes later, 1 ml of blood was collected from inferior vena cava of
each mouse under Nembutal anesthesia. 1251 radioactivity in blood was
then measured using a gamma counter (Alola ARC300). Furthermore, to
examine the in vivo stability of various liposome complexes, the serum of
each blood was re-chromatographed using Sephadex G-50. In all cases, most
of radioactivity was observed in high-molecular-weight void fractions.
Various liposome complexes had stability also in vivo. In addition, each
amount of radioactivity that had been transferred from the intestinal
tract into blood is represented by the proportion of radioactivity per ml
of blood (% dose/ml blood) with respect to the total radioactivity
administered.

[0430]As a result, the types and binding densities of sugar chains, which
were optimum for oral administration, were determined.

(Summary of Examples 22 and 23)

[0431]In vivo assay results of the targeting properties (tropism) of the
delivery vehicles prepared based on the rolling model were faithfully
reproduced in vitro. Furthermore, a more appropriate delivery vehicle
could be selected by taking the binding density of a sugar chain into
consideration. Such targeting properties remained unchanged regardless if
the relevant delivery vehicle contained a drug or did not contain a drug.

Example 24

Preparation of Vitamin A-Encapsulated Liposome, and Determination and
Storage Stability of Encapsulated Drug

[0432]Next, nutritional elements were delivered using delivery vehicles
that had been revealed by the rolling model to be appropriate. A liposome
was prepared using cholic acid dialysis. Specifically,
dipalmitoylphosphatidylcholine, cholesterol, dicetylphosphate,
ganglioside, and dipalmitoylphosphatidylethanol amine were mixed at a
molar ratio of 35:40:5:15:5 so that the total amount of lipid was 45.6
mg. 46.9 mg of sodium cholate was added to the mixture and then the
resultant was dissolved in 3 ml of a chloroform/methanol solution. The
solution was evaporated and then the precipitate was dried in vacuum,
thereby obtaining a lipid membrane. The thus obtained lipid membrane was
suspended in 3 ml of a TAPS buffered saline solution (pH8.4) and then the
resultant was ultrasonicated, so that 10 ml of a transparent micelle
suspension was obtained. Vitamin A that had been completely dissolved in
a TAPS buffer solution (pH8.4) to a concentration of 3 mg/1 ml was slowly
added dropwise to the micelle suspension while agitating the suspension.
After homogeneously mixing the suspension, the vitamin A-containing
micelle suspension was subjected to ultrafiltration using a PM10 membrane
(Amicon Co., U.S.A.) and a TAPS buffered saline solution (pH8.4) so that
10 ml of a homogeneous vitamin A-encapsulated liposome particle
suspension was prepared. The particle diameter and zeta potential of the
vitamin A-encapsulated liposome particles in the thus obtained
physiological saline suspension (37° C.) were measured using a
zeta potential-particle diameter-molecular weight measurement apparatus
(Model Nano ZS, Malvern Instruments Ltd., UK). As a result, the particle
diameter ranged from 50 nm to 350 nm and the zeta potential ranged from
-30 mV to -10 mV. The amount of the drug encapsulated in the liposome was
found by measuring the absorbance at 260 nm. It was revealed that vitamin
A was encapsulated at a concentration of approximately 280 μg/ml. The
vitamin A-encapsulated liposome remained stable without undergoing
precipitation and aggregation even after 1 year of storage in a
refrigerator.

[0433]Liposomes capable of achieving delivery to desired sites could be
prepared using such liposome.

[0436]Galectin 1 is expressed in skeletal muscle, neurons, the kidney, the
placenta, and the thymus gland. Galectin 2 is expressed in liver tumors.
Galectin 3 is expressed in activated macrophages, eosinophils,
neutrophils, mast cells, the small intestine, and epithelial and sensory
neurons of respiratory organs. Galectin 4 is expressed in the epithelia
of the intestine and oral cavity. Galectin 5 is expressed in erythrocytes
and reticulum cells. Galectin 6 is expressed in intestinal epithelia.
Galectin 7 is expressed in keratinocytes. Galectin 8 is expressed in the
lungs, the liver, the kidney, the heart, and the brain. Galectin 9 is
expressed in the liver, the small intestine, the kidney, lymphoid
tissues, the lungs, cardiac muscles, and skeletal muscles. They can be
useful as indicators for delivery vehicle for use in delivery to these
organs.

[0437]A mannose-6-phosphate receptor is distributed in the trans-Golgi
network of each cell, so that it is useful as an indicator for a delivery
vehicle for use in delivery to these cell sites.

[0438]Calnexin is distributed in the endoplasmic reticulum, so that it is
useful as an indicator for a delivery vehicle for use in delivery to the
cell site.

[0439]Calreticulin is distributed in the endoplasmic reticulum so that it
is useful as an indicator for a delivery vehicle for use in delivery to
these cell sites.

[0440]ERGIC-53 is distributed in the endoplasmic reticulum to cis-Golgi
regions so that it is useful as an indicator for a delivery vehicle for
use in delivery to these cell sites.

[0441]VIP36 is distributed in the endoplasmic reticulum to cell membrane
regions so that it is useful as an indicator for a delivery vehicle for
use in delivery to these cell sites.

[0442]Siglec1 (sialoadhesin) is distributed in macrophages so that it is
useful as an indicator for a delivery vehicle to be used for delivery to
these sites.

[0443]Siglec2 (CD22) is distributed in lymphocytes (B cells) so that it is
useful as an indicator for a delivery vehicle to be used for delivery to
these sites.

[0444]Siglec3 (CD33) is distributed in myeloid cells so that it is useful
as an indicator for a delivery vehicle to be used for delivery to these
sites.

[0445]Siglec4a (MAG) is present in the peripheral nerve so that it is
useful as an indicator for a delivery vehicle to be used for delivery to
these sites.

[0446]Siglec5 (myelin protein) is present in monocytes so that it is
useful as an indicator for a delivery vehicle to be used for delivery to
these sites.

[0447]N-CAM is distributed in the peripheral nerve, so that it is useful
as an indicator for a delivery vehicle to be used for delivery to these
sites.

[0448]Po (intercellular adhesion factor that is present on mammalian
peripheral myelin and mature Schwann cells) is distributed in the
peripheral nerve so that it is useful as an indicator for a delivery
vehicle to be used for delivery to these sites.

[0449]L-selectin is distributed in leukocytes so that it is useful as an
indicator for a delivery vehicle to be used for delivery to these sites.

[0450]P-selectin is present in vascular endothelial cells so that it is
useful as an indicator for a delivery vehicle to be used for delivery to
these sites.

[0451]A mannose binding protein is present in lymphocytes (natural killer
cells) so that it is useful as an indicator for a delivery vehicle to be
used for delivery to these sites.

[0452]An asialo glycoprotein receptor is distributed in the liver so that
it is useful as an indicator for a delivery vehicle to be used for
delivery to these sites.

[0453]A macrophage mannose receptor is distributed in macrophages so that
it is useful as an indicator for a delivery vehicle to be used for
delivery to these sites.

[0454]Antithrombin (blood coagulation factor) is present in blood so that
it is useful as an indicator for a delivery vehicle to be used for
delivery to these sites.

[0455]FGF is distributed in blood so that it is useful as an indicator for
a delivery vehicle to be used for delivery to these sites.

[0456]Interleukin2 (IL-2) is distributed in blood so that it is useful as
an indicator for a delivery vehicle to be used for delivery to these
sites.

[0457]Interleukin1α (IL-1α) is distributed in blood so that it
is useful as an indicator for a delivery vehicle to be used for delivery
to these sites.

[0458]Interleukin1β (IL-1β) is distributed in blood so that it
is useful as an indicator for a delivery vehicle to be used for delivery
to these sites.

[0459]Interleukin3 (IL-3) is distributed in blood so that it is useful as
an indicator for a delivery vehicle to be used for delivery to these
sites.

[0460]Interleukin6 (IL-6) is distributed in blood so that it is useful as
an indicator for a delivery vehicle to be used for delivery to these
sites.

[0461]Interleukin7 (IL-7) is distributed in blood so that it is useful as
an indicator for delivery vehicle to be used for delivery to these sites.

[0462]Tumor necrosis factor α (TNF-α) is distributed in blood
so that it is useful as an indicator for a delivery vehicle to be used
for delivery to these sites.

[0463]If these cell surface molecules are subjected to an experiment
according to Example 7 and the result is used as a rolling model,
delivery vehicle preferable in vivo in corresponding organs and sites can
be searched for.

[0464]The relationships between cell surface molecules and organs referred
in the Examples can be described as follows. Studies concerning various
lectins (sugar chain-recognizing proteins including C-type lectin such as
selectin, DC-SIGN, DC-SGNR, collectin, or a mannose binding protein,
1-type lectin such as siglec, P-type lectin such as a mannose-6-phosphate
receptor, R-type lectin, L-type lectin, M-type lectin, and galectin) as
receptors existing on cell membrane surfaces and the like of various
tissues in vivo have been advanced. Sugar chains having various molecular
structures are attracting attention as new DDS ligands.

[0465]Regarding the relationships between cell surface molecules and
organs, for example, cell surface molecules the expression of which in
human tissues has been revealed are as listed below:

[0466]Regarding the relationships between cell surface molecules and
disease tissues, expression of E-selectin, P-selectin, and the like in
general inflammatory diseases (e.g., encephalitis, chorioretinitis,
pneumonia, hepatitis, and arthritis) and diseases that cause inflammation
successively (e.g., malignant tumor, rheumatism, cerebral infarction,
diabetes, and Alzheimer disease) is being elucidated. Furthermore,
expression of E-selectin in cancerous tissues has been reported. Most
about the relationships between cell surface molecules and organs remains
unknown and the future elucidation thereof is expected.

[0467]When animal lectins are classified based on their primary
structures, they are classified into the following 14 types of family,
for example:

[0468]As the above relationships between a wide variety of animal lectins
and organs or diseases have been revealed, it is predicted that the
delivery vehicle (e.g., sugar chain-modified liposome) of the present
invention will be more useful for treatment and diagnosis of diseases and
will be able to be applied to wider application fields. Moreover, also
for the purpose of elucidation of the biological significance of various
animal lectins, the delivery vehicle of the present invention is useful
as a reagent for research or the like.

Example 26

Preparation of Ganglioside-Free Sugar Chain-Modified Liposome

(1. Preparation of Liposome)

[0469]A liposome was prepared by the techniques of the previous report
(Yamazaki, N., Kodama, M. and Gabius, H.-J. (1994) Methods Enzymol. 242,
56-65) using improved cholic acid dialysis. Specifically,
dipalmitoylphosphatidylcholine, cholesterol, dicetylphosphate, and
dipalmitoylphosphatidylethanol amine were mixed so that the total amount
of lipid was 45.6 mg. 46.9 mg of sodium cholate was added to the mixture
and then the resultant was dissolved in 3 ml of a chloroform/methanol
solution. The solution was evaporated and then the precipitate was dried
in vacuum, thereby obtaining a lipid membrane. The thus obtained lipid
membrane was suspended in 3 ml of a TAPS buffer solution (pH8.4) and then
the resultant was ultrasonicated, so that a transparent micelle
suspension was obtained. Furthermore, the micelle suspension was
subjected to ultrafiltration using a PM10 membrane (Amicon Co., U.S.A.)
and a PBS buffer solution (pH7.2). Thus 10 ml of a homogeneous liposome
(average particle diameter: 100 nm) was prepared.

(2. Hydrophilization of Liposome Lipid Membrane Surface

[0470]10 ml of the liposome solution prepared in 1 above was subjected to
ultrafiltration using an XM300 membrane (Amicon Co., U.S.A.) and a CBS
buffer solution (pH8.5) and the pH of the solution was adjusted to pH8.5.
Next, 10 ml of a cross-linking reagent bis(sulfosuccinimidyl)suberate
(BS3; Pierce Co., U.S.A.) was added, followed by 2 hours of agitation at
25° C. Subsequently, the solution was further agitated overnight
at 7° C. so as to complete the chemical binding reaction between
lipid dipalmitoylphosphatidylethanol amine on the liposome membrane and
BS3. The liposome solution was then subjected to ultrafiltration using an
XM300 membrane and a CBS buffer solution (pH8.5). Next, 40 mg of
tris(hydroxymethyl)aminomethane dissolved in 1 ml of a CBS buffer
solution (pH8.5) was added to 10 ml of the liposome solution, followed by
2 hours of agitation at 25° C. The solution was then agitated
overnight at 7° C., so as to complete the chemical binding
reaction between BS3 bound to the lipids on the liposome membrane and
tris(hydroxymethyl)aminomethane. Thus, the hydroxy group of
tris(hydroxymethyl)aminomethane was coordinated on the lipid
dipalmitoylphosphatidylethanol amine of the liposome membrane, so that
the liposome membrane surface was hydrated and hydrophilized.

[0471]Human serum albumin (HSA) was bound to the liposome membrane surface
by techniques similar to those in Example 3. As a result, human serum
albumin (HSA) was not bound to the liposome membrane surface. Through
evaluation of the thus obtained delivery vehicle using the rolling model,
it could be determined whether or not the delivery vehicle was
appropriate vehicle.

Example 27

Delivery Vehicle Other than Sugar Chain-Modified Liposome

[0472]Next, it was verified whether or not the rolling model can be
applied to a delivery vehicle other than the sugar chain-modified
liposome.

[0474]It could be determined whether or not the thus obtained delivery
vehicles were appropriate via evaluation based on the rolling model.

[0475]As described above, the present invention is exemplified using the
preferred embodiments of the present invention. The present invention
should not be interpreted as being limited to the embodiments. It is
understood that the scope of the present invention should be interpreted
based only on the claims. It is understood that persons skilled in the
art can implement the present invention within the scope equivalent to
the specific preferred embodiments of the present invention based on
descriptions of the present invention and technical commonsense. It is
also understood that patents, patent applications, and publications cited
herein are incorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

[0476]The present invention has usefulness such that a delivery vehicle
capable of delivering a substance to a target delivery site can be freely
designed. Therefore, the present invention provides a delivery vehicle
(e.g., sugar chain-modified liposome) in which a substance (e.g., a drug
or a gene) is encapsulated and usefulness (e.g., treatment, diagnosis,
prevention, and research) relating thereto.